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Biomedical subjects

I Tsoneva

Publications and source records attributed to I Tsoneva.

12 recordsLinked to original sources
PubMed2006

Electrochemotherapy of Mycosis fungoides by interferon-alpha.

Eight patients with 29 lesions of histologically verified 1st stage of Mycosis fungoides were successfully treated by electrochemotherapy with interferon-alpha. For this purpose 8 biphasic pulses were used, each of 50+50 micros duration with 900 micros interpulse intervals, resulting in a burst of 7.1 ms total duration. Compared to the traditional monoimmunotherapy with interferon-alpha applied three times weekly for a total of 4 weeks, the electrochemotherapy was very efficient. Complete response (CR) was observed in 25 (86%) of the 29 treated lesions by single-act electrochemotherapy with interferon-alpha. At the end of the 12-month period, all 29 lesions showed 100% complete response (CR). New lesions for a period of 12 months were not observed. The expected mechanism involved in multiple cytotoxic action of interferon-alpha could be the local increased concentration in the tumour and prolongation of the time of its action after the application of pulses.

Angiogenesis Inhibitors↗
PubMed2004

Induction of apoptosis by electrotransfer of positively charged proteins as Cytochrome C and Histone H1 into cells.

Cytochrome C (Cyt. C) is a mitochondrial protein inducing apoptosis when it is accumulated in the cytosol by a currently unknown mechanism, but regulated by the bcl-2 family of proteins. The linker Histone H1 is another basic protein with highly conservative structure, composition, and equal molecular weight, not changed during the evolution. An attempt was made to understand better the apoptotic processes by electroloading of leukemic cells, such as K562, HL-60, and SKW3, and human lymphocytes with positively charged proteins, such as Cyt. C, Histone H1, and methylated BSA albumin (mBSA). The triggering apoptotic processes followed by MTT test, FACS analysis, and DNA fragmentation after the electrotransfer of these proteins into the cells were observed. Histone H1 and mBSA induce the release of Cyt. C from rat liver mitochondria. Cytochrome C release was higher when mitochondria were in "high-energy" state. It is supposed that release of Cyt. C from mitochondria is due to the mechanical rupture of the outer mitochondrial membrane, rich in negatively charged groups, predominately due to cardiolipin. The reason for the morphological rupture of the outer mitochondial membrane could be the rigidification and segregation of the membrane and the destroyed membrane asymmetries of both monolayers in the presence of positively charged proteins at higher linear charges such as Histone H1. We suggested that Histone H1, at a given moment of activated signaling for apoptosis, could be not transported to the nucleus and could lead to the release of Cyt. C from the mitochondria in the cytoplasm. It is temping to speculate that Histone H1 has other physiological extranuclear functions involved in apoptosis.

Animals↗
PubMed2002

An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles.

A new approach for direct optical microscopy observation of polyanion interactions with bilayers of giant cationic liposomes (GUVs) was suggested. Polyanions as DNA, dextran sulfate (DS), heparin (H) and polyacrylic acids (PA) were locally delivered by a micropipette to a part of a giant unilamellar vesicle membrane. The phenomena were directly observed under optical microscope. GUVs, about 100 micro m in diameter, formed of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine (Sph), were prepared by electroformation. The effects of water-soluble molecules with high negative linear charge density as dextran sulfate (DS), heparin (H) polyacrylic acids (PA) and adenosine-5'-triphosphoric acid (ATP) were compared with those of DNAs. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis-like membrane morphology transformation due to the DNA/lipid membrane local interactions was observed. The DS, H and PA induced membrane topology transformations similar to those of the DNAs, while ATP did not cause any detectable ones. The endocytosis mechanism involves the formation of ordered domains in the GUV membrane where some surface and charge asymmetries between the two membrane monolayers were created. The sizes of created polyanionic/cationic membrane domains depend on the form, length and elasticity of the adsorbed highly charged molecules. Endosome-including capacities of polyanionic molecules depend heavily on the high linear negative charge at a certain length. An original method for direct studying of the DNA/membrane interactions in autoadaptable giant liposome system imitating biological membrane interactions was forwarded. The model observations could also help for understanding events associated with cationic liposome/DNA complex formation in gene transfer processes.

Acrylic Resins↗
PubMed2000

Are the stainless steel electrodes inert?

The electrodes used for electroporation, electrofusion, and electrotransfection, as well as in electrochemotherapy should not interact with the object and release electrolysis products. We studied how far the stainless steel electrodes fulfil these requirements. The results showed that in the case of the exponential electric pulses with amplitude of 3 kV cm(-1) and time constant of 0.5 ms, the stainless steel electrodes released ferrous ions to a concentration of several micromoles per liter. We determined experimentally the dependence of the amount of the released ferrous ions on the pulse parameters and on the ionic strength of the medium. It was shown that the released ferrous ions should not be ignored. They could be of importance for the bleomycin electrochemotherapy.

Electrodes↗
PubMed1999

DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles.

We suggest a novel approach for direct optical microscopy observation of DNA interaction with the bilayers of giant cationic liposomes. Giant unilamellar vesicles, about 100 microns in diameter, made of phosphatidyl-cholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. "Short" DNAs (oligonucleotide 21 b and calf thymus 250 bp) were locally injected by micropipette to a part of the giant unilamellar vesicle (GUV) membrane. DNAs were injected native, as well as marked with a fluorescent dye. The resulting membrane topology transformations were monitored in phase contrast, while DNA distribution was followed in fluorescence. We observed DNA-induced endocytosis due to the DNA/lipid membrane local interactions and complex formation. A characteristic minimum concentration (Cendo) of D-erythrosphingosine (Sph+) in the GUV membrane was necessary for the endocytic phenomenon to occur. Below Cendo, only lateral adhesions between neighboring vesicles were observed upon DNA local addition. Cendo depends on the type of zwitterionic (phosphocholine) lipid used, being about 10 mol% for DPhPC/Sph+ GUVs and about 20 mol% for SOPC/Sph+ or eggPC/Sph+ GUVs. The characteristic sizes and shapes of the resulting endosomes depend on the kind of DNA, and initial GUV membrane tension. When the fluorescent DNA marker dye was injected after the DNA/lipid local interaction and complex formation, no fluorescence was detected. This observation could be explained if one assumes that the DNA is protected by lipids in the DNA/lipid complex, thereby inaccessible for the dye molecules. We suggest a possible mechanism for DNA/lipid membrane interaction involving DNA encapsulation within an inverted micelle included in the lipid membrane. Our model observations could help in understanding events associated with the interaction of DNA with biological membranes, as well as cationic liposomes/DNA complex formation in gene transfer processes.

Animals↗
PubMed1999

Interactions of DNA with giant liposomes.

DNA interactions with the bilayers of cationic liposomes were studied using a novel model experiment: DNAs were locally injected by a micropipette to a part of a giant unilamellar vesicle. The resulting phenomena were directly observed in optical microscope. Giant unilamellar vesicles (GUVs), about 100 microm in diameter, made of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. The effects of DNAs of different length were tested: (i) 'short' DNAs-oligonucleotide 21b, and calf thymus 250 bp; (ii) 'long' DNAs-plasmid DNAs in super coil or liner form (between 2.7 and 8.0 kbp). DNAs were injected native, as well as marked with the fluorescent dye Hoechst. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis was observed due to the DNA/lipid membrane local interactions for all DNAs tested. Some of the DNA in the formed complex was associated with the induced endosomes, and some of it remained spread over the 'mother' GUV membrane for all DNAs tested, except for the longest one--the linear plasmid of 8 kbp. The last remained at the 'mother' GUV membrane and was not transported with the induced endosomes to the internal GUV space. Possible mechanisms for DNA/lipid membrane interaction were suggested. One of them involves DNA encapsulation within an inverted micelle included in the lipid membrane. The model observations could help in understanding events associated with interaction of DNA with biological membranes, as well as cationic liposomes/DNA complexes formation in gene transfer processes.

Animals↗
PubMed1997

Sphingosine-mediated electroporative DNA transfer through lipid bilayers.

When the cationic sphingosine is present in planar lipid bilayers, the adsorption of pDNA is enhanced and the electroporatability of the bilayer is facilitated. Furthermore, pDNA, adsorbed to lipid bilayers composed of diphytanoyl lecithin and D-sphingosine causes electroporative channel-like events of conductance 1 to 7 pS, provided the voltage polarity is correct and the voltage is high enough, Vm > or = +30 mV. The data suggest electrotransport of pDNA through the bilayer, mediated by transient complexes between DNA and the lipids in the pore edges of elongated, electropercolated hydrophilic pore zones. The results are the basis for the optimisation of electroporative transfer of DNA or oligonucleotides to cells and tissue.

DNA↗
PubMed1996

Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation.

Detailed kinetic data suggest that the direct transfer of plasmid DNA (YEp 351, 5.6 kbp, supercoiled, Mr approximately 3.5 x 10(6)) by membrane electroporation of yeast cells (Saccharomyces cerevisiae, strain AH 215) is mainly due to electrodiffusive processes. The rate-limiting step for the cell transformation, however, is a bimolecular DNA-binding interaction in the cell interior. Both the adsorption of DNA, directly measured with [32P]dCTP DNA, and the number of transformants are collinearly enhanced with increasing total concentrations [Dt] and [Cat] of DNA and of calcium, respectively. At [Cat] = 1 mM, the half-saturation or equilibrium constant is KD = 15 +/- 1 nM at 293 K (20 degrees C). The optimal transformation frequency is TFopt = 4.1 +/- 0.4 X 10(-5) if a single exponential pulse of initial field strength E0 = 4 kV cm-1 and decay time constant tauE = 45 ms is applied at [Dt] = 2.7 nM and 10(8) cells in 0.1 ml. The dependence of TF on [Cat] yields the equilibrium constants KCazero = 1.8 +/- 0.2 mM (in the absence of DNA) and K'Ca (at 2.7 nM DNA), comparable with and derived from electrophoresis data. In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event. At Eo = 4.0 kV cm-1 and T = 293 K, the flow coefficient of DNA through the porous membrane patches is Kto = 7.0 +/- 0.7 x 10(3)S-1 and the electrodiffusion of DNA is approximately 10 times more effective than simple diffusion: D/D0 approximately 10.3. The mean radius of these pores is rp = 0.39 +/- 0.05 nm, and the mean number of pores per cell (of size ø approximately 5.5 microns) is Np = 2.2 +/- 0.2 x 10(4). The maximal membrane area that is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023% of the total cell surface. The surface penetration is followed either by additional electrodiffusive or by passive (after-field) diffusive translocation of the inserted DNA into the cell interior. For practical purposes of optimal transformation efficiency, 1 mM calcium is necessary for sufficient DNA binding and the relatively long pulse duration of 20-40 ms is required to achieve efficient electrodiffusive transport across the cell wall and into the outer surface of electroporated cell membrane patches.

Adsorption↗
PubMed1994

Dip patch clamp currents suggest electrodiffusive transport of the polyelectrolyte DNA through lipid bilayers.

Planar lipid bilayers formed from monolayers of diphytanoyl lecithin (DPhL) were found to interact with plasmid DNA (5.6 kbp; M(r) = 3.7 x 10(6)) leading to an increase in the conductance of the membrane. The association of DNA with a lipid bilayer greatly facilitates the transport of the small ions of the main salt KCl. The appearance of long-lived current levels, for instance, of 27.6 pA at Vm = +60 mV membrane voltage, where the actual contact (adsorption) is electrophoretically enhanced, suggests a locally conductive DNA/lipid interaction zone where parts of the DNA strand may be transiently inserted in the bilayer, leaving other parts of the DNA probably protruding out from the outer surface of the bilayer. At Vm = -60 mV, where DNA can be electrophoretically moved away from the membrane, the membrane current is practically zero. This current asymmetry is initially also observed at higher voltages, for instance at 200 mV. However, if the voltage sign (Vm = +200 mV) is changed after a transient positive current (approximately 15 pA) was observed, there is also now (at Vm = -200 mV) a finite negative current at the negative membrane voltage. Thus, it appears that at Vm = +200 mV the adsorbed parts of the polyelectrolyte DNA are not only transiently inserted in, but actually also electrophoretically pulled through, the porous zones onto the other membrane side leaving the bilayer structure basically intact. These data provide direct electric evidence for the electrophoretic transport of a highly charged and hydrated macromolecule, probably together with the associated gegen-ions, through the thin hydrophobic film of the lipid bilayer.

Diffusion↗
PubMed1989

Electrofusion of fragile mutants of Saccharomyces cerevisiae.

This communication presents experimental evidence that intact fragile (osmotic sensitive) yeasts can be electrofused and give viable hybrids. The yield increases with one order of magnitude for electrofusion of intact fragile yeasts with protoplasts of non-fragile ones. The yield of viable hybrids, obtained by electrofusion of protoplasts of fragile and non-fragile yeasts, is one order of magnitude higher than the yield from protoplasts of non-fragile yeasts. The destabilized cell wall and plasma membrane of the mutant yeasts could be a possible explanation for this phenomenon.

Electricity↗
PubMedDate not supplied

Kinetics of calcium-induced fusion of cell-size liposomes with monolayers in solutions of different osmolarity.

The effects of osmolarity, calcium concentration and cell-size liposomes in the subphase on the surface tension of phospholipid monolayers were investigated. The monolayers were spread from chloroform solutions of phosphatidic acid at air/water solution interface. The liposomes (of average diameter 3 micron) were formed from phosphatidic acid/egg lecithin (1:2) mixtures in water or 0.1 M water solutions of sucrose. For this system there were critical concentrations of calcium ions to produce a large reduction of the monolayer surface tension. The threshold calcium concentrations depended upon the sucrose concentration in the subphase. Without sucrose the threshold calcium concentration was 8 mM, while for isoosmotic sucrose solutions (0.1/0.1 M in/out of liposome) it was 14 mM. It sharply increased to 28 mM CaCl2 at sucrose concentration difference across the liposome membrane 0.02 M and decreased to 26 mM, 19 mM, and 18 mM with further increase of that difference to 0.04 M, 0.06 M, and 0.08 M, respectively. The rate of monolayer surface tension decrease was measured as a function of time at 30 mM CaCl2 and different sucrose concentrations in the subphase solution. The initial rates at first decreased with increasing the osmotic pressure and after that they increased. The minimum occurred at sucrose concentration gradient across the liposome membrane 0.02 M, i.e., at the point of maximum threshold calcium concentration required for large decrease of the monolayer surface tension. These facts may be explained by recent theories of dynamics of adhesion, instability and fusion of membranes modeled as thin films.

Calcium↗