Calcium permeability of synaptosomes induced by alpha-latrotoxin

The paper deals with characteristics of ionic alpha-latrotoxin-induced permeability of rat brain synaptosomes. It has been shown that the addition of alpha-latrotoxin to synaptosomes in the Ca2+-containing media resulted in an extensive and rapid uptake of 45Ca2+ in synaptosomes. alpha -Latrotoxin was not able to enhance the 22Na+ and 86Rb+ uptake or efflux in the Ca2+-containing and Ca2+- and Mg2+-free media. The dye di-O-C3 was used to monitor the membrane potential changes as a consequence of alpha-latrotoxin treatment of synaptosomes. It has been found that alpha-latrotoxin increased synaptosomal fluorescence in the Ca2+-containing media, but failed to induce any increase of fluorescence in Ca2+- and Mg2+-free media. It has been also shown that the calcium uptake induced by alpha-latrotoxin depends on free calcium concentration in synaptosomes. Toxin-induced calcium flows are shown to be of the vector character.     

Latrotoxin-induced fusion of liposomes with bilayer phospholipid membranes

Liposomes containing amphotericin B as ionophoric marker were used to investigate the fusion of bilayer phospholipid membranes with liposomes. It was found that latrotoxin isolated from black widow spider venom induced the fusion of liposomes with planar bilayer when liposomes and latrotoxin were administered at opposite sides of the membrane.     

Antigenic similarity between cytoplasmic tetrodotoxin-sensitive protein and neuronal membrane proteins

Membrane proteins with a molecular weight of 290, 180, and 55 kDa were isolated using immunosorbent attached to sepharose and rabbit antibodies to cytoplasmic tetrodotoxin-sensitive protein from beef brain gray matter. A technique used for research into voltage-dependent sodium channels was applied to reconstruction of these proteins and investigation of toxin-dependent sodium flows through the lipoprotein membrane. Findings are interpreted as evidence of the similarity between cytoplasmic tetrodotoxin-sensitive protein and that of sodium channels at the cell membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev; A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 485–489, July–August, 1989.     

Resistance of liposomes with different physico-chemical characteristics to the effect of plasma

The chemical composition, liquid content sign and value of charge as well as structure and size of lipid vesicles are studied for the effect they exert on the liposome permeability for 22Na+ in the presence of human blood plasma. The rate of the isotope outlet from the electroneutral lecithin liposomes is determined by the size of vesicles and the quantity of phospholipid bilayers in their membrane. The presence either of a negative or a positive charge on the surface of the liposome membrane has no essential effect on the outlet rate of the radioactive marker. Introduction of different amounts of cholesterol or sphingomyelin into the liposome composition decreases considerably the lipid vesicle permeability and an increase in the liquid content of their membranes due to the temperature elevation is accompanied by a sharp rise in the isotope outlet rate. A conclusion is drawn on the possibility to control the outlet rate of the liposome content in the presence of blood plasma.     

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3′ ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson–Crick base pair at the end. Mismatched duplexes with a 3′ dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5′ exonuclease activity of the enzyme.     

New Pyrimidine Cyclonucleosides with Hydrogenated Aglycones: Synthesis and X-Ray Structures

Abstract

Acid catalysed transformations of (6S)-6,5′-anhydro-6-hydroxy-1-(2′,3′-O-isopropylidene-β-D-ribofuranosyl)hexahydropyrimidine-2-thione are studied. (6R)-6,2′-anhydro-6-hydroxy-1-(α-D-ribofuranosyl)hexahydropyrimidine-2-thione was formed as a thermodynamically stable product. Two intermediates, (6S)-6,5′-anhydro-6-hydroxy-1-(β-D-ribofuranosyl)hexahydropyrimidine-2-thione and 6-hydroxy-1-(D-ribosyl)hexahydropyrimidine-2-thione and products of cleavage of glycosidic bond were identified in the reaction mixtures. Results of X-ray structural determination of the synthesised nucleosides are presented.     

Transport of Rb+ via activated sodium channels of clone N 18 phi 1 neuroblastoma cells

A study was made of the Rb+ transport via activated sodium channels of clone N 18 phi 1 neuroblastoma cells cultured in the Eagle medium with 10% bovine serum. The time of population doubling was about 10 h. The cell differentiation was induced by adding bromdeoxyuridine in a concentration of 1-4 10(-5) M. The cells contained 172 +/- 12 and 340 +/- 35 micrograms of protein per 10(6) cells at the logarithmic growth phase and in differentiated state, respectively. It is shown that veratrin produced a 1.3-fold increase in the rate of 86Rb+ removal from undifferentiated cells and 2.5-fold increase in that from differentiated cells. Tetrodotoxin removed completely the effect of veratrin. A conclusion is made on the presence of a new clone of fast sodium channels in cell membranes.     

Design of Tripeptides as a Competitive Inhibitor for HMG-CoA Reductase

International Journal of Peptide Research and Therapeutics - This study presents a simple approach in design of tripeptides as a competitive inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase...     

Serine Protease EspP from Enterohemorrhagic Escherichia Coli Is Sufficient to Induce Shiga Toxin Macropinocytosis in Intestinal Epithelium

Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells’ basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.     

Delivery and processing of exogenous double-stranded DNA in mouse CD34 + hematopoietic progenitor cells and their cell cycle changes upon combined treatment with cyclophosphamide and double-stranded DNA

We previously reported that fragments of exogenous double-stranded DNA can be internalized by mouse bone marrow cells without any transfection. Our present analysis shows that only 2% of bone marrow cells take up the fragments of extracellular exogenous DNA. Of these, ~ 45% of the cells correspond to CD34 + hematopoietic stem cells. Taking into account that CD34 + stem cells constituted 2.5% of the total cell population in the bone marrow samples analyzed, these data indicate that as much as 40% of CD34 + cells readily internalize fragments of extracellular exogenous DNA. This suggests that internalization of fragmented dsDNA is a general feature of poorly differentiated cells, in particular CD34 + bone marrow cells.