Vol. 3 No. 4 December 1998

Volume 3 (1998) pp 365-374
Title INTERACTION OF ANNEXIN VI WITH MEMBRANES. REGULATION BY ATP IN VITRO
Authors J. Bandorowicz-Pikuła1*, M. Danieluk1, A. Wrzosek2, R. Buś1 and S. Pikuła1
Abstract Porcine liver annexin VI (AnxVI) has recently been described to bind in vitro ATP. The binding of nucleotide to protein is accompanied by modulation of AnxVI function, such as its' interaction with F-actin and membranes. In the present report, we show that ATP modulates AnxVI-driven aggregation of phosphatidylserine (PS) liposomes. In addition, we provide evidence using circular dichroism (CD) that the interaction of AnxVI with ATP evokes changes in secondary structure of the protein. The functional implications of these changes are also discussed.
Address and Contact Information Departments of 1Cellular and 2Muscle Biochemistry, Nencki Institute of Experimental Biology,
3 Pasteur Street, 02-093 Warsaw, Poland
*Corresponding author. Fax: (+4822) 822 5342; E-mail: bandor@nencki.gov.pl
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 375-378
Title MULTIPLE EXTRACTION METHOD FOR DNA, RNA AND NUCLEAR PROTEIN ISOLATION FROM PERIPHERAL BLOOD
Authors A. E. Ferry and B. S. Pace
Abstract Obtaining adequate samples for nucleic acid or protein analysis from a limited number of cells can be a difficult task. The steps for isolation of DNA, cytoplasmic RNA and nuclear proteins from mononuclear cells collected from a single peripheral blood sample are outlined below. A previously described technique for rapid isolation of nuclear proteins was modified to acquire RNA and DNA of sufficient quantity and quality to perform analyses at the molecular level without altering the quality of protein extracted. This approach is applicable for use with peripheral blood mononuclear cells, primary cultures and immortalized cell lines.
Address and Contact Information University of South Alabama, Department of Structural and Cellular Biology Mobile, AL 36688, U.S.A.
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 379-388
Title POLYMORPHISMS OF HUMAN IL-1b AND IL-1 ANTAGONIST GENES AND THEIR INFORMATIVITY FOR THE ASSOCIATION STUDIES
Authors H.W. Witas, W. Młynarski and R. Sychowski
Abstract Several genetic and immunological studies showed that interleukin-1 (IL-1) is involved in pathogenesis of immune-related disorders e.g. type 1 diabetes, autoimmune thyroiditis or inflammatory bowel disease. Association studies performed till now are rather inconclusive, possibly because investigated markers chosen were not enough informative. The aim of the present study is to elucidate how informative for association screening is the occurrence of known polymorphisms located within genes encoding IL 1b (IL-1B) and IL 1 antagonist (IL 1RN) in Polish population. The following substitutions were analyzed by PCR based techniques: TŽC within the promotor region, GŽA in intron 4 and CŽT in exon 5 of IL-1B gene as well as TŽC in exon 2, TŽC in exon 4 and the polymorphism of variable number of tandem repeats (VNTR) of 86 base pairs within intron 2 of IL-1RN gene. Obtained results proved the occurrence of all studied polymorphisms in Polish population. Combination of haplotypes related to three novel IL 1B gene dimorphisms reveals that PIC value 0.77 (polymorphic information contents) is similar to that of microsatellite markers. Thus, it is possible that the combination of revealed polymorphisms makes together new marker applicable for the genetic association studies more sensitive then markers previously used.
Address and Contact Information Molecular Biology Unit, 2nd Clinic of Children Diseases,
Institute of Pediatrics, Medical University of Łódź, Sporna 36/50, 91-738 Łódź, Poland
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 389-392
Title STABILIZATION OF LYOPHILIZATION-SENSITIVE ANTIBODY-ENZYME CONJUGATES
Authors D. Arora, A. K. Chauhan and N.Khanna
Abstract A simple method for stabilization of lyophilization or freezing/thawing sensitive enzyme-antibody conjugates is described. The method involves soaking of these reagents directly onto the placebo homoepathic sugar tablets followed by a brief air drying. This solid dry formulation is stable at elevated room temperature and is very convenient for storing or shipping small aliquots of these lyophilzation sensitive reagents
Address and Contact Information International Centre for Genetic Engineering and Biotechnology,
P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi - 110 067, INDIA
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 393-402
Title THE MEMBRANE-CYTOSKELETON INTERFACE CONTROLS PLATELET SHAPE CHANGE
Authors J. H. Hartwig1*, K. Barkalow1+, A. Azim1, J. Mcgrath1, K. Tolias2 and C. Carpenter2
Abstract Platelets respond to stimuli by rapidly changing from discs into active forms having lamellipodia and filopodia. Actin filament fragmentation and assembly are essential for platelet shape change. Actin assembly is regulated by an intracellular signaling pathway that involves calcium and the activation of small GTPases and phosphatidylinositol kinases (PI-kinases). Polyphosphoinositides (ppIs), the final products of the PI-kinases, directly interact with a special class of actin associated proteins, the barbed end capping proteins, to initiate actin filament assembly in activated cells.
Address and Contact Information 1Hematology Division, Brigham and Women's Hospital, Boston, MA and 2 Division of Signal Transduction, Department of Medicine, Beth Israel Hospital and Department of Cell Biology, Harvard Medical School, Boston, MA 02115.
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 403-411
Title NEW THERAPEUTIC APPROACHES TO SICKLE CELL DISEASE: TARGETING RBC MEMBRANE OXIDATIVE DAMAGE
Authors S.R. Goodman, B.S. Pace and A. Shartava
Abstract In this brief review, we discuss evidence leading to the conclusion that diminished levels of reduced glutathione combined with increased oxygen radical production leads to oxidative damage to membrane proteins in sickle cell disease erythrocytes. Among these oxidatively damaged proteins are K+-channels (Gardos channel and K+-Cl--channel) and beta-actin. Oxidative damage to the K+-channels leads to K+ leakage and H20 loss from light density reversibly sickled cells (RSCs). The resulting dense RSCs are primarily sickled in shape due to increased [HbS] and increased polymerization. The oxidation of beta-actin converts the dense RSCs to dense irreversibly sickled cells (ISCs) as explained in our two step model. Furthermore, we discuss recent in vitro evidence that N-acetylcysteine (NAC) can block the formation of dense cells and ISCs by protecting the K+-channels and beta-actin respectively from oxidative damage. Finally we describe an ongoing Phase II human trial to determine whether NAC can also lower dense cells and ISCs in vivo and, if so, result in fewer painful vasooclusive episodes.
Address and Contact Information University of South Alabama College of Medicine, Department of Structural and Cellular Biology,
USA Comprehensive Sickle Cell Center, Mobile, Alabama 36688, U.S.A.
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 413-422
Title NEWER FACETS OF SICKLE CELL ADHESIVENESS
Authors S. B. Shohet1, B. J.-M. Thevenin1, I. W. Sherman2 and U. Von Andrian3
Abstract To further explore the finding that peptides derived from specifically exposed regions of band 3 inhibited sickle cell adhesiveness in vitro, we examined the effects of such peptides with human sickle cells in the mouse microcirculation in vivo. Human red cells were loaded with the fluorescent dye BCECF, and infused into the arterial circulation of anaesthetized mice. The microcirculation of several tissues was examined in a specially designed warm-stage microscope with stroboscopic epi-fluorescent illumination. Adhesive events were video-recorded and quantified after peptide infusion and after treatment of the mice with various biological modifiers. An adhesive index (AI), proportionate to the number of adhesive events per number of cells entering a vessel, was calculated. Sickle cells (SS) were found to be more adhesive than normal cells particularly in small vessels such as post-capillary venules of the cremaster muscle (AI 2.3 vs 0.5). Moreover, endothelium activation substantially increased SS adhesiveness (e.g., AI 8.3 after pretreatment with platelet activating factor (PAF), 5.8 with tumor necrosis factor, and 8.7 with thrombin-activated mouse platelets). A band 3 peptide which blocked SS adhesiveness in vitro inhibited much of the increased adhesiveness of SS cells (e.g., in the PAF-treated mouse AI 2.8 vs 8.3, vs pretreatment control 2.3). No inhibitory effect was seen with a control peptide with the same composition but a scrambled sequence. These preliminary findings suggest that the inhibitory activity of specific band 3 sequences on SS adhesiveness is retained in vivo and extend the previous in vitro demonstration of an adhesive role for these exposed band 3 sequences. In addition, they imply a substantial role for endothelial and/or platelet activation in the adhesiveness of sickle cells in vivo. Finally, they demonstrate the utility of direct visualization of the mouse microcirculation for studying the adhesiveness of human cells.
Address and Contact Information 1University of California, San Francisco, 2University of California, Riverside, 3Center for Blood Research, Harvard Medical School
[Rozmiar: 1332 bajtów]


Volume 3 (1998) pp 423-433
Title MODULATED HUMAN GLOBIN GENE EXPRESSION: ROLE FOR ANTISENSE EXPRESSION VECTORS
Authors L. Xu1 and B. S. Pace2
Abstract Several approaches have been explored to prevent polymerization of sickle hemoglobin in erythrocytes. We tested the ability of a mammalian expression vector carrying a b gene antisense cDNA fragment to block b gene expression. The antisense expression vector was stably transfected into HS2gb stable mouse erythroleukemia cell lines producing human g and b globin chains. By day 14 there was an average 24% decrease and 12% increase in b and g globin mRNA levels respectively. We observed a loss of b gene inhibition by day 42. This study suggests that a b globin antisense cDNA expression vector may be an alternative gene therapy strategy to decrease sickle hemoglobin levels in patient with sickle cell disease.
Address and Contact Information University of South Alabama,
1Department of Biology, 2Department of Structural and Cellular Biology, Mobile, AL 36688, U.S.A.
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 435-441
Title DEHYDRATED HEREDITARY STOMATOCYTOSIS REVISITED
Authors J. Delaunay1,2, S. Grootenboer2, P.O. Schischmanoff3, T. Cynober1, G. Tchernia 1,2, J.-P. Dommergues4, M. Bost5, G Stewart6, S. Perrotta7, M. Carella8, P. Gasparini8 and A. Iolascon9
Abstract Dehydrated hereditary stomatocytosis (DHS), also designated hereditary xerocytosis, is a chronic congenital haemolytic anaemia, underlain by abnormal transmembrane fluxes of Na+ and K+. The intracellular concentrations of Na+ and K+ are increased and decreased, respectively, though to various degrees. Stomatocytes and other abnormally shaped red cells appear on smears. The osmotic gradient ektacytometric curve is shifted leftward. Cell dehydration, macrocytosis and/or high reticulocyte counts are noted. Electrophoresis of membrane proteins is unremarkable and, in particular, stomatin is present. The inheritance pattern of DHS is invariably dominant, although the severity and even the nature of the symptoms may vary within some particular kindreds. DHS may be associated with hereditary pseudohyperkalaemia (HPHK) and/or perinatal oedema (PO), as has been recently discovered. HPHK, per se, is a lifelong asymptomatic trait characterized by a dramatic elevation of kalaemia when blood is allowed to stand for a few hours at room temperature. Its inheritance pattern is dominant. Modulation of expression within families has not been mentionned to date. PO one is dealing with here has the unique property of spontaneously receding after birth. We have screened a number of families with DHS in order to define the single or multiple facets of the clinical phenotype. We found cases with DHS alone, HPHK alone, DHS + HPHK, DHS + PO, and a case with DHS + HPHK + PO. One bias was that HPHK was not necessarily searched for at the adequate temperature in any particular kindred. Another bias was that PO, having been slim and transient, could recede prior to birth in some cases. Taken together, the clinical genetics led us to postulate that the multiple presentation of DHS might nonetheless stem from mutations all gathered in the gene encoding, presumably, an ion transporter or channel. Based on the study of a large Irish family with isolated DHS, preliminary results indicated that the responsible locus maps to chromosome 16 (16q23-qter).
Address and Contact Information 1Service d Hematologie, d Immunologie et de Cytogenetique,
2INSERM U473,
3Service de Biochimie I, et
4Service de Pediatrie, Hospital de Bicotre, 94275 Le Kremlin-Bicotre, France;
5Laboratoire d Hematologie, Grenoble, France ;
6Department of Medicine, The Rayne Institute, London, UK;
7Dipartimenti di Pediatria, Napoli, Italy;
8Servizio di Genetica Medica, IRCCS-CSS, San Giovani Rotondo, Italy;
9Dipartimento dell Etŕ Evolutiva, Universitŕ di Bari, Italy.
Phone: ++ (33) 1 45 21 20 16; Fax: ++ (33) 1 45 2128 47; email: delaunay@kb.inserm.fr
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 443-448
Title CYLINDRICAL SHAPES OF RED BLOOD CELL MICROEXOVESICLES
Authors A. Iglic1, V. Kralj-Iglic2, P. Peterlin2 and H. Hagerstrand3
Abstract Cylindrical microexovesicles were induced in human erythrocytes by echinocytic amphiphile dodecyl maltoside. It is suggested that the effect of the curvature deviator is relevant for the stability of the observed cylindrical microexovesicle shapes.
Address and Contact Information 1Laboratory of Applied Physics, Faculty of Electrical Engineering, University of Ljubljana, SI-1000, Ljubljana, Slovenia, 2Institute of Biophysics, Medical Faculty, University of Ljubljana, SI-1000, Ljubljana, Slovenia and 3Department of Biology, Abo Akademi University, FIN-20520, Abo/Turku, Finland
[Rozmiar: 1332 bajtów]

Volume 3 (1998) pp 449-463
Title SKELETON - BILAYER INTERACTION AND THE SHAPE OF RED BLOOD CELLS
Authors S. Svetina
Abstract The shapes of red blood cells with fluid interior depend on the properties of their membranes. The red blood cell membrane is composed of a bilayer, containing integral proteins, and the underlying membrane skeleton, which are attached to each other through different specific and nonspecific linkages. A short account is given of theories of shape formation based on the description of the membrane as a single- or multi-layered elastic continuum. Existing experimental evidence is then outlined which indicates that some relevant shape determining factors reside in the specific properties of different red blood cell integral and skeletal proteins. In this context a theoretical approach is introduced, based on the chemical equilibria established by the interacting skeletal and integral proteins. A simple prototype model is analyzed, taking into consideration that the binding between integral membrane proteins and the skeleton depends on membrane curvature. The essential parameters of the model are the gross value of the corresponding interaction energy and the coefficient measuring the dependence of this energy on membrane curvature. The effects of these parameters on RBC shapes and on the lateral mobility of integral membrane proteins are demonstrated.
Address and Contact Information Institute of Biophysics, Medical Faculty, University of Ljubljana and Institute J. Stefan, Ljubljana, Slovenia
[Rozmiar: 1332 bajtów]