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 |

|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
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