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Biomedical subjects

V K Lishko

Publications and source records attributed to V K Lishko.

At least 19 recordsLinked to original sources
PubMed2001

The alternatively spliced alpha(E)C domain of human fibrinogen-420 is a novel ligand for leukocyte integrins alpha(M)beta(2) and alpha(X)beta(2).

The interaction of human plasma fibrinogen with leukocyte integrins alpha(M)beta(2) (CD11b/CD18, Mac-1) and alpha(X)beta(2) (CD11c/CD18, p150,95) is an important component of the inflammatory response. Previously, it was demonstrated that binding of fibrinogen to these integrins is mediated by gammaC, the globular C-terminal domain of the gamma chain. In this study, evidence was found of another fibrinogen domain that can serve as a ligand for the 2 leukocyte integrins: alpha(E)C, a homologous domain that extends the alpha chains in a recently discovered subclass of fibrinogen known as fibrinogen-420. Recombinant alpha(E)C supported strong adhesion and migration of cells expressing alpha(M)beta(2) and alpha(X)beta(2), including nonactivated and activated U937 and THP-1 monocytoid cells, and neutrophils. Cells transfected with complementary DNA for these integrins also bound alpha(E)C. The specificity of interaction was substantiated by inhibition of cell adhesion with antibodies against alpha(M), alpha(X), and beta(2) subunits. Also, neutrophil inhibitory factor, a specific inhibitor of alpha(M)beta(2) and alpha(X)beta(2) function, efficiently blocked cell adhesion to alpha(E)C. In alpha(M)beta(2) and alpha(X)beta(2), the I domain is the binding site for alpha(E)C, since alpha(E)C bound to recombinant alpha(M) I and alpha(X)I domains in a dose-dependent and saturable manner. Synthetic peptides that duplicated sequences gamma190 to 202 and gamma377 to 395, previously considered putative binding sites in gammaC, effectively inhibited alpha(M)beta(2)- and alpha(X)beta(2)-mediated adhesion to alpha(E)C, suggesting that recognition of alpha(E)C by the I domain involves structural features in common with those of gammaC. These findings identify alpha(E)C as a second domain in fibrinogen-420 that binds alpha(M)beta(2) and alpha(X)beta(2) and can mediate leukocyte adhesion and migration.

Alternative Splicing↗
PubMed1996

Probing the structure-function relationship of alpha-latrotoxin-formed channels with antibodies and pronase.

The major toxic component of black widow spider (Latrodectus mactans tredecimguttatus) venom, alpha-latrotoxin, is known to form ionic channels in different membranes. In order to probe the extramembrane domains of alpha-latrotoxin molecule, alpha-latrotoxin channels in planar lipid membrane were treated with antibodies to latrotoxin or with pronase added to different sides of the membrane. It was found that antibody addition to the same side as the toxin (cis) decreased channel conductance only at positive potentials across the membrane. In contrast, trans side addition of antibodies changed the channel conductance at both positive and negative potentials: at positive potential conductance first slightly increased then decreased by more then 50%; at negative potential it decreased much more quickly, to only about 20% of the initial value. No dependence on membrane potential was found for pronase treatment of incorporated channels. For both cis and trans application of pronase, channel selectivity for Ca2+, Mg2+, Ba2+ and K+, Na+, Li+ ions did not change significantly but Cd2+ block was decreased. Trans pronase treatment also resulted in some rectification of I/V curves and an increase in channel conductance. We interpret these findings as evidence that alpha-latrotoxin channel has protruding parts on both sides of the membrane and that its conformation in the membrane depends on membrane potential.

Animals↗
PubMed1994

[Binding of secreted protein mRNA translation products with artificial phospholipid vesicles].

Translocation of eucaryotic secretory proteins across the phospholipid membrane containing no protein components has been studied. The level of translocation was found to depend critically on the physico-chemical properties of the membranes. It was found also that eucaryotic secretory proteins can pass through the phospholipid bilayer by both co- and post-translational interactions.

Animals↗
PubMed1993

Therapeutic tumor-specific cell cycle block induced by methionine starvation in vivo.

The ability to induce a specific cell cycle block selectively in the tumor could have many uses in chemotherapy. In the present study we have achieved this goal of inducing a tumor-specific cell cycle block in vivo by depriving Yoshida sarcoma-bearing nude mice of dietary methionine. Further, we demonstrate that methionine depletion also causes the tumor to eventually regress. The antitumor effect of methionine depletion resulted in the extended survival of the tumor-bearing mice. The mice on the methionine-deprived diets maintained their body weight for the time period studied, indicating that tumor regression was not a function of body weight loss. The data reported here support future experiments utilizing methionine depletion as a target for tumor-selective cell cycle-dependent therapy.

Animals↗
PubMed1993

The preparation of endotoxin-free L-methionine-alpha-deamino-gamma-mercaptomethane-lyase (L-methioninase) from Pseudomonas putida.

Many types of human and animal tumors have an absolute requirement for methionine. This requirement can be satisfied by homocysteine only in normal cells and tissues. Therefore, methionine may be an important target in cancer therapy. To attack this target we have purified endotoxin-free methioninase from Pseudomonas putida by a novel and simple procedure. This procedure involves (1) a heat step of the cell extract at 60 degrees C for 8 min, (2) DEAE-Toyopearl ion-exchange chromatography, (3) DEAE-Sephadex A50 ion-exchange gel filtration chromatography, and (4) affinity chromatography on Acticlean to remove the endotoxin bound to the enzyme. The yield for this purification was up to 80%. The methioninase has four subunits of approximate molecular weight 43 kDa. This is the first methodology for methioninase that allows rapid purification with high yield and separation from endotoxin suitable for in vivo efficacy testing against methionine-dependent tumors in animal models.

Carbon-Sulfur Lyases↗
PubMed1992

Latrotoxin-like properties of a protein from brain.

In bovine brain cortex cytoplasm we have identified a soluble protein (L-protein) of M(r) approximately 90 kDa interacting with polyclonal antibodies to alpha-latrotoxin. The L-protein forms potential-dependent and cation-selective ion channels in BLM, which are blocked by Cd2+. The fusogenic activity of the L-protein was demonstrated on liposomes. We have arrived at the conclusion that the action mechanisms of the L-protein and alpha-latrotoxin are similar.

Animals↗
PubMed1992

Potential-dependent alpha-latrotoxin interaction with black lipid membranes.

The influence of membrane potential on alpha-latrotoxin insertion into bilayer lipid membranes (BLM) has been investigated. It was found that positive potentials cis to toxin application stimulated the formation of channels in the bilayer. A two-step model of latrotoxin/membrane interaction is put forward to explain these data. In the first step, latrotoxin irreversibly binds to the bilayer without forming conductive structures. The second step of the process represents rapid insertion of the protein molecule into the bilayer with the formation of the conducting channel. We imagine the driving force for this process to be the interaction of charged groups in the toxin molecule with the electric field applied across the BLM. Our results are compared with known data on the interaction of LTX with synaptosomal membranes.

Electric Conductivity↗
PubMed1992

[Prevention of postischemic lesions of the myocardium using liposomes].

The experiments on dogs showed that 60-min blood flow restriction in the left coronary artery branch resulted in pumping and contractile heart dysfunctions. The removal of the blood flow barrier caused reinforcement of the above dysfunctions. The administration of 50 mg/kg liposome prior to reperfusion improved pumping and contractile heart functions and allowed maintenance of stable hemodynamics during the reperfusion.

Animals↗
PubMed1992

[Functioning of latrotoxin channels during pH changes].

Using the fluorescent probe BCECP, the pH dependence of Ca2+ transport in synaptosomes along alpha-latrotoxin-formed channels, was studied. It was found that the pH value in synaptosomes is equivalent to 7.16 +/- 0.09. Acidification or alkalinization of the intracellular medium by 0.1-0.3 pH units had no appreciable influence on the Ca2+ influx along latrotoxin-formed channels. Alteration of external pH caused a parallel shift in the cytoplasmic pH in the synaptosomes. The pH decrease in the external medium down to 6.0 caused the inhibition of Ca2+ fluxes along latrotoxin-formed channels. Dissipation of the proton gradient by high concentrations of KCl in the presence of nigericin decreased the latrotoxin ability to form ionic channels without any loss in the activity of the preformed channels. The influx of bivalent cations along latrotoxin-formed channels led to alkalinization of the synaptosomal cytoplasm to pH of the external medium. This pH change did not depend on the presence of Na+ in the external medium and was blocked by cadmium.

Animals↗
PubMed1990

Fusion of negatively charged phospholipid vesicles by alpha-latrotoxin.

alpha-Latrotoxin-induced fusion of liposomes has been described using large unilamellar vesicles composed of phosphatidylcholine/phosphatidylethanolamine/cardiolipin at a molar ratio of 2:3:5. Vesicle fusion was monitored by terbium/dipicolinic acid assay as well as by fluorescence energy transfer measurement. The enhancement of the fusogenic effect of LTX by low concentrations (0.1-3 mM) of CaCl2 has been demonstrated. The efficiency of other divalent cations on the LTX fusogenic activity was shown to decrease in the sequence Ca greater than Cd greater than Sr greater than Mg greater than Ba. LTX-induced fusion was accompanied by the increase of vesicle size measured by laser correlation spectroscopy. It is concluded that fusogenic action of LTX may be involved in its effect on synaptic apparatus.

Arthropod Venoms↗
PubMed1990

[Internalization and degradation of latrotoxin bound to rat brain synaptosomes].

The accessibility to trypsin of 125I-labeled latrotoxin bound to rat brain synaptosomes was investigated. It was shown that latrotoxin bound to synaptosomes in the cold can be practically completely removed by trypsin treatment. The resistance of latrotoxin to proteolysis increases during its incubation with synaptosomes (37 degrees C). Concanavalin A (10(-6) M) decreases toxin binding by 30%, but fully prevents internalization (incorporation). Moreover, latrotoxin is not incorporated into synaptosomal membrane fragments irrespective of duration and temperature of incubation. Latrotoxin incorporated into synaptosomal membranes undergoes degradation by endogenous proteases resulting in the formation of TCA-soluble products.

Animals↗
PubMed1990

[Latrotoxin channels. Permeability for divalent cations].

The dependence of Ca2+ transport in synaptosomes along the channels formed by alpha-latrotoxin on [Ca2+]in and the feasibility of transport along these channels of other bivalent cations were studied. It was found that the concentration dependence of Ca2+ influx is nonlinear and is described by the Michaelis-Menten kinetics (Km = 1.07 +/- 0.19 mM). Mg2+, Ba2+, Sr2+, Mn2+ and Co2+ competitively inhibited the Ca2+ influx via latrotoxin channels. Studies with the use of the fluorescent Ca2+ probes, Quin-2 and Fura-2, revealed that these cations can also penetrate inside synaptosomes via latrotoxin channels. The bivalent cation influx via latrotoxin channels caused a decrease of the membrane potential of synaptosomes. The similarity of properties of latrotoxin and endogenous Ca2+ channels is discussed.

Aminoquinolines↗
PubMed1990

[The physiological mechanisms of the antihypoxic action of the liposomes].

The effect of phospholipids introduced into the vascular bed in the form of liposomes, was studied in pats in conditions of breathing with hypoxic gas mixture containing 7% of oxygen in nitrogen. The liposomes were shown to improve the oxygen diffusion from the blood into tissues and from air to the blood. In the result of this, the degree of the tissue hypoxia is considerably reduced, the process of peroxide oxidation of lipids is inhibited, and the efficacy of external respiration and gas exchange is increased.

Animals↗
PubMed1990

[Biological effect of liposomes in hypoxic conditions of various etiologies].

The antihypoxic and antioxidative effects of phosphatidyl choline liposomes have been studied in hypoxic hypoxia, pneumonia, and acute blood loss. It was demonstrated that the body tolerance of persisting hypoxia increased on liposome administration due to elimination of lactate-acidosis, inhibition of lipid peroxidation and higher rate of oxygen diffusion through the biological barriers. The antihypoxic properties of the vesicles are determined by their remedial effect on the key mechanisms responsible for the development of hypoxic organ damage.

Animals↗