Vol. 6 No. 3 September 2001

Volume 6 (2001) pp 571-585
Title STUDIES OF THE ERYTHROCYTE SPECTRIN TETRAMERIZATION REGION
Authors Sunghyouk Park1, Shahila Mehboob2, Bing-Hao Luo2, Michael Hurtuk2, M. E. Johnson1 and L. W.-M. Fung2
Abstract Human erythrocyte spectrin dimers associate at the N-terminal region of a-spectrin (aN) and the C-terminal region of b-spectrin (bC) to form tetramers. We have prepared model peptides to study the tetramerization region. Based on phasing information obtained from enzyme digests, we prepared spectrin fragments consisting of the first 156 amino-acid residues and the first 368 amino-acid residues of a-spectrin (Spa1-156 and Spa1-368, respectively), and found that both peptides associate with a b-spectrin model peptide, with an affinity similar to that found in ab dimer tetramerization. Spin label EPR studies show that the region consisting of residues 21-46 in a-spectrin is helical even in the absence of its b-partner. Multi-dimensional nuclear magnetic resonance studies of samples with and without a spin label attached to residue 154 show that Spa1-156 consists of four helices, with the first helix unassociated with the remaining three helices, which bundle to form a triple helical coiled coil bundle. A comparison of the structures of erythrocyte spectrin with other published structures of Drosophila and chicken brain spectrin is discussed. Circular dichroism studies show that the lone helix in Spa1-156 associates with helices in the b-peptide to form a coiled coil bundle. Based on NMR and CD results, we suggest that the helices in Spa1-156 exhibit a looser (frayed) conformation, and that the helices convert to a tighter conformation upon association with its b-partner. This suggestion does not rule out possible conversion of a non-structured conformation to a structured conformation in various parts of the molecule upon association. Spectrin mutations at residues 28 and 45 of a-spectrin have been found in patients with hereditary elliptocytosis. NMR studies were also carried out on Spa1-156R28S, Spa1- 156R45S and Spa1-156R45T. A comparison of the structures of Spa1-156 and Spa1-156R28S, Spa1-156R45S and Spa1-156R45T is discussed.
Address and Contact Information 1Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, and
2Department of Chemistry, Loyola University of Chicago, Chicago, IL 60626
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Volume 6 (2001) pp 587-591
Title REGENERATION OF CUPHEA TOLUCANA PEYR. IN IN VITRO CULTURE
Authors Zbigniew Przybecki1, Jan Olejniczak2 and Elżbieta Adamska2
Abstract In order to regenerate Cuphea tolucana from hypocotyl, cotyledon and root explants, a solid culture and 8 hormone combinations were used. Only the root explants did not react to any of the media. On most of the media, the other explants formed shoots, roots or callus, or their reaction was more complex. The regeneration probably occurred via direct organogenesis. The regenerants displayed a wide variety of morphological characteristics. However, their offspring did not show any differences from plants which had not undergone culture.
Address and Contact Information 1Department of Plant Genetics, Breeding and Plant Biotechnology, Warsaw University of Agriculture, Nowoursynowska 166, 02-787 Warszawa, Poland,
2Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, 60-479 Poznan, Poland
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Volume 6 (2001) pp 593-606
Title NEW INSIGHTS INTO RED CELL NETWORK STRUCTURE, ELASTICITY, AND SPECTRIN UNFOLDING
Authors Dennis E. Discher and Philippe Carl
Abstract New insights into red cell network structure, elasticity, and spectrin unfolding - a current review. The red cell membrane's well-recognized ability to withstand the stresses of circulation clearly has its origins in various levels of spectrin-actin network structure. We highlight recently obtained insights into this sub-structure and also briefly explain the implications to membrane components that interact with the network. Novel insights into the resilience of this cytoskeleton are being provided by experiments that range from atomic force microscopy (AFM) tests of single, unfoldable spectrin chains to micropatterned photobleaching of a pipette-deformed network. Continued progress in atomic level structure determinations of non-erythroid spectrin and related repeats are further complemented by theoretical efforts - computational approaches most notably - that have begun to correlate molecular scale aspects of structure with micro-mechanical measures. All of this recent activity in the biophysics of red cell structure-function challenges and refines some of the most basic tenets in cell membrane response.
Address and Contact Information Institute for Medicine and Engineering, School of Eng. and Applied Science, and Structural Biology Program - The Wistar Institute, Biophysical Engineering Lab, 112 Towne Bldg. University of Pennsylvania, Philadelphia, PA 19104-6315, USA, tel (215) - 898 - 4809, fax (215) - 573 - 6334
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Volume 6 (2001) pp 607-636
Title SPECTRIN UBIQUITINATION AND OXIDATIVE STRESS: POTENTIAL ROLES IN BLOOD AND NEUROLOGICAL DISORDERS
Authors Jose Sangerman, David Kakhniashvili, Aprile Brown, Archil Shartavaand Steven R. Goodman*
Abstract This review covers the observations that erythrocyte spectrin has a E2 ubiquitin conjugating enzymatic activity that allows it to transfer ubiquitin to a target site in the α-spectrin repeats 20/21. The position of this ubiquitination site suggests that ubiquitination may regulate αβ spectrin heterodimer nucleation, spectrin-4.1-actin ternary complex formation, and adducin stimulated spectrin-actin attachment in the mature erythrocyte. In sickle cells, which contain altered redox status (high GSSG/GSH ratio), ubiquitin attachment to the E2 and target sites in α-spectrin is greatly diminished. We propose that this attenuated ubiquitination of spectrin may be due to glutathiolation of the E2 active site cysteine leading to diminished ubiquitin- spectrin adduct and conjugate formation. Furthermore we propose that lack of ubiquitin-spectrin complex formation leads to dysregulation of the membrane skeleton in mature SS erythrocytes and may diminish spectrin turnover in SS erythropoietic cells via the ubiquitin proteasome machinery. In hippocampal neurons, spectrin is the major ubiquitinated protein and a component of the cytoplasmic ubiquitinated inclusions observed in AlzheimerÎŁs and ParkinsonÎŁs diseases. The two primary neuronal spectrin isoforms: αSpIΣ*/βSpIΣ2 and αSpIIΣ1/βSpIIΣ1 are both ubiquitinated. Future work will resolve whether neuronal spectrins also contain E2-ubiquitin conjugating activity and the molecular basis for formation of ubiquitinated inclusions in neurological disorders.
Address and Contact Information Department of Cell Biology and Neuroscience and USA Comprehensive Sickle Cell Center, University of South Alabama College of Medicine, Mobile AL 36688, USA, Department of Molecular and Cell Biology, University of Texas at Dallas, Dallas, TX, USA
*Corresponding author: tel.972-883-4872, e-mail: sgoodmn@utdallas.edu
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Volume 6 (2001) pp 637-648
Title ENZYME-LINKED IMMUNOSORBENT ASSAY IN SCREENING OF LEUKEMIA-ASSOCIATED NUCLEAR PROTEINS
Authors Małgorzata Rogalińska1, Jerzy Błoński2, Tadeusz Robak2 and Zofia M. Kiliańska1*
Abstract Our previous data revealed some diversities in electrophoretic characteristics of nuclear fraction proteins isolated from peripheral blood mononuclear cells of B-cell chronic lymphocytic leukemia (B-CLL) patients and healthy donors. Two electrophoretically-specific nuclear non-histones in the molecular mass zone of 38/39 and 44/46 kDa of leukemic mononuclear cells were used as immunogens to produce rabbit antisera. The Western blot analysis indicated that both nuclear components are expressed only in mononuclear cells isolated from peripheral blood of B-CLL patients, but not in those isolated from the blood of healthy donors. For further investigations of nuclear fraction from normal and B-CLL mononuclear cells, an enzyme-linked immunosorbent assay (ELISA) was used. The results obtained by ELISA with the antisera raised against both electrophoretically-specific B-CLL nuclear polypeptides revealed a different extend of cross-reactivity of nuclear fraction preparations isolated from normal cells and those isolated from leukemic ones. We noticed that nuclear fraction preparations which originated from leukemic mononuclear cells are much more reactive than normal ones with both antisera (at a broad range of antisera dilutions).
Address and Contact Information 1Department of Cytobiochemistry, University of Lodz , Banacha 12/16, 90-237 Lodz , Poland,
2Department of Hematology, Medical University of Lodz , Paderewskiego 4, 93-513 Lodz , Poland
* Corresponding author - e-mail: zkilian@biol.uni.lodz.pl
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Volume 6 (2001) pp 649-675
Title IMPACT OF FOUR ANTIMUTAGENS ON APOPTOSIS IN GENOTOXICALLY DAMAGED LYMPHOCYTES IN VITRO
Authors Kazimierz Gąsiorowski1*, Barbara Brokos1, Anna Kulma2, Antoni Ogorzałek3 and Katarzyna Skorkowska1
Abstract An antimutagenic activity of fluphenazine, todralazine, anthocyanins and alkylresorcinols was established in a battery of short-term cytogenetic tests. One of the possible mechanisms of their antimutagenic action could be an increase in apoptotic elimination of heavily-damaged cells from a culture. In this paper we provide data on quantitative estimation of the antimutagensµ impact on apoptosis in lymphocyte cultures exposed in the G0-phase to genotoxic agents: hydrogen peroxide (0.2mM, 20 min.) or benzo[a]pyrene (40 ����90 min.), and then cultured for 36 hrs in the presence of a lectin (PHA-M, 1% v/v) and each of the tested antimutagens. Apoptosis was estimated by means of microscopic examination of cell smears stained with a mixture of fluorochromes (ethidium bromide/acridine orange) as well as of the results of DNA separation with the field inversion gel electrophoresis. By microscopic examination we assessed that the frequencies of cells exhibiting morphological features of apoptosis considerably increased in the cultures containing the antimutagens. The FIGE separation of DNA from those cultures proved that the DNA content in the 30-50 kb domain was markedly elevated, as compared with the control cultures that did not contain antimutagens. It was established in the regression analysis that the apoptosis-enhancing effect significantly depended on the concentration of each tested antimutagen in a culture medium. However, marked differences of apoptosis-enhancig potency were noticed among the four antimutagens. The multicriterial analysis proved that the apoptosis-enhancing effects of fluphenazine and also, to a smaller extent, by alkylresorcinols, were many times stronger than those of anthocyanins and of todralazine. The results suggest that the enhancement of apoptosis by fluphenazine and by alkylresorcinols can explain a major part of their antimutagenic activity, whereas in the case of anthocyanins and of todralazine other mechanisms of antimutagenic action should be sought for.
Address and Contact Information 1Wrocław Medical University, Department of Basic Medical Sciences, Kochanowskiego 14, 51- 601 Wrocław, Poland,
2University of Wrocław, Department of Genetic Biochemistry, Przybyszewskiego 63/77, 51-148 Wrocław, Poland,
3University of Wrocław, Department of Zoology, Sienkiewicza 21, 50-335 Wrocław, Poland
*Corresponding author, fax: (+4871) 3479211,e-mail: kaz@basmed.am.wroc.pl
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Volume 6 (2001) pp 677-690
Title MECHANICAL AND FUNCTIONAL ASPECTS OF MEMBRANE SKELETONS
Authors Sasa Svetina1,2, Bojan Bozic1, Jure Derganc1 and Bostjan Zeks1,2
Abstract Membrane skeletons can be characterized as cytoskeletal structures lying parallel to the bilayer part of cellular and organelle membranes. Typical examples are spectrin network and actin-myosin cortex. We approach the problem of elucidating the function of membrane skeletons by theoretically analyzing mechanical models of the cellular behavior. Membranes of different physical and chemical properties are considered. In erythrocytes and some organelles membrane bilayers are smooth and simply underlaid or overlaid by membrane skeletons. It is argued that there the role of a membrane skeleton is, either, to keep the membrane composition laterally homogeneous as it is in the case of the erythrocyte, or, that it is involved in the processes of the lateral separation of integral membrane proteins as it is happening in the case of some intermediate steps of the vesicular membrane trafficking. In the second type of membranes the bilayer part is ruffled and folded, and there the membrane skeletons play a role in the determination of the cortical tension. Here we explore in more detail the mechanical behavior of a cell with such properties of its boundary. The shape transformations are described which occur under the influence (i) of different external forces, i.e., when an originally spherical cell is aspirated into the micropipette or when such a cell is adsorbed on a flat surface, and (ii) of different internal forces on the cell boundary exerted by the cytoskeletal elements.
Address and Contact Information 1Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Slovenia and 2 J. Stefan Institute, Ljubljana, Slovenia
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Volume 6 (2001) pp 691-702
Title THE POSTSYNAPTIC SPECTRIN/4.1 MEMBRANE PROTEIN 'ACCUMULATION MACHINE'
Authors Anthony J. Baines, Lisa Keating, Gareth W. Phillips and Catherine Scott
Abstract An important aspect of the function of the membrane-associated cytoskeleton has been suggested to be to trap and retain selected transmembrane proteins at points on the cell surface specified by cell adhesion molecules. In the process, cell adhesion molecules are cross-linked to each other, and so junctional complexes are strengthened. In this short review, we will discuss recent advances in understanding the role of this "accumulation machine" in postsynaptic structures. Function in the brain depends on correct ordering of synaptic intercellular junctions, and in particular the recruitment of receptors and other apparatus of the signalling system to postsynaptic membranes. Spectrin has long been known to be a component of postsynaptic densities, and recent advances in molecular cloning indicate that b spectrins at PSDs are all "long" C-terminal isoforms characterised by pleckstrin homology domains. Isoforms of protein 4.1 are also present at synapses. All four 4.1 proteins are represented in PSD preparations, but it is 4.1R that is most enriched in PSDs. 4.1R binds to several proteins enriched in PSDs, including the characteristic PSD intermediate filament, a-internexin. Both 4.1 and spectrin interact with ionotropic glutamate receptors (AMPA and NMDA receptors, respectively): 4.1 stabilises AMPA receptors on the cell surface. By linking these receptors to the cytoskeletal and cell adhesion molecules that specify glutamatergic synapses, the membrane protein accumulation machine is suggested to direct the formation of postsynaptic signalling complexes.
Address and Contact Information Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK

Volume 6 (2001) pp 703-720
Title MECHANOPROTECTION OF THE PLASMA MEMBRANE IN NEURONS AND OTHER NON-ERYTHROID CELLS BY THE SPECTRIN-BASED MEMBRANE SKELETON
Authors Catherine E. Morris
Abstract Though the cytomechanics of spectrin have been explored only for erythrocytes, it is thought that the spectrin skeleton acts universally to support the otherwise mechanically vulnerable cell surface bilayer. Evidence for this role is beginning to accumulate and is reviewed here. Compared to that for erythrocytes, cells whose simplicity facilitates biophysical approaches, the evidence is indirect. One way that membrane skeleton/bilayer interactions have been probed is via the behavior of mechanosusceptible ion channels -- channel whose gating is perturbed by abnormally high bilayer tension. These initially unresponsive channels become progressively more mechanoresponsive as stretch and chemical reagents damage the membrane skeleton. The straightforward implication is that the intact membrane skeleton is mechanoprotective. In non-erythroid cells there is continual trafficking of bilayer to and from the plasma membrane. Some of the traffic involves spectrin- lined vacuolar membrane. Several lines of evidence suggest that when neurons elongate and remodel their neurites, membrane skeleton-based mechanoprotection allows the dynamic vacuoles and the plasma membrane to participate in mechanosensitive surface area expansion and retrieval.
Address and Contact Information Neurosciences, Ottawa Health Research Institute, Ottawa Hospital, 725 Parkdale Ave, Ottawa, Ontario, Canada K1Y 4K9