Degree Of Permeabilization Research Articles

Liposomal Permeabilization Assay to Study the Functional Interactions of the BCL-2 Family.

Apoptosis, a form of programmed cell death that is important for development and homeostasis, is regulated by the BCL-2 family of proteins. Over twenty BCL-2 family members have been classified in three groups based on structural homology and function. The multidomain antiapoptotic proteins promote survival, whereas the multidomain and the BH3-only proapoptotic members induce cell death. Because the interaction among the BCL-2 family members occurs primarily at the mitochondrial outer membrane, biochemical assays using artificial liposomes have been developed to study the functional relationship between these proteins. The liposomal permeabilization assay is a cell-free system that relies on the ability of multidomain pro-apoptotic members to promote membrane permeabilization upon activation. By encapsulating a fluorophore and a quencher into liposomes, the degree of permeabilization can be quantified by the increase in fluorescence intensity as the fluorophore and quencher dissociate. The liposomal permeabilization assay has been used to delineate interactions among BCL-2 family members as well as to characterize peptides, small molecules, and lipids that modulate the function of BCL-2 family of proteins. Here, we describe in detail the permeabilization of liposomes induced by the interaction between BAX and BH3-only activator tBID.

A Voronoi Interface approach to cell aggregate electropermeabilization

We present a Voronoi Interface approach to the study of cell electropermeabilization. We consider the nonlinear electropermeabilization model of Poignard et al. [20], which takes into account the jump in the voltage potential across cells' membrane. The jump condition is imposed in a sharp manner, using the Voronoi Interface Method of Guittet et al. [14], while adaptive Quad/Oc-tree grids are employed to automatically refine near the cells boundary for increased accuracy. Numerical results are provided to illustrate the accuracy of the methods. We also carry out simulations in three spatial dimensions to investigate the influence of shadowing and of the cells shape on the degree of permeabilization.

Effect ofBacillus cereusHemolysin II on Hepatocyte Cells

We investigated the efficiency of increasing the permeability (permeabilization) of cell membranes in primary liver cells by Bacillus cereus hemolysin II. An assessment of the degree of permeabilization was car ried out by measuring the fluorescence intensity of various low molecular weight dyes, which enter through pores into hepatocyte cells cultivated with hemolysin. We uncovered a high efficacy of hemolysin HlyII action on hepatocyte cell walls, which exceeded the effect of nonionic detergent, digitonin, which is commonly employed for pore formation in various cell membranes. Our results also point to the reversibility of membrane permeabilization in primary hepatocytes. The data obtained in this study can be utilized for assessments of pore-forming activity, in studies of hepatic mechanisms of action, and also the determination of the liver toxicity for different low molecular weight drugs.

Permeabilization and lysis induced by bacteriocins and its effect on aldehyde formation by Lactococcus lactis

Permeabilization induced by lacticin 3147, lactococcins A, B and M, enterocin AS-48 and nisin, bacteriocins described as cell membrane-pore forming and lytic agents, enhanced in all cases aldehyde formation by Lactococcus lactis IFPL730. Nevertheless, the conversion of isoleucine into 2-methylbutyraldehyde depended not only on the degree of permeabilization but also on the bacteriocin that caused the cell membrane damage. The highest values of 2-methylbutyraldehyde corresponded to cell suspensions containing lacticin 3147 and lactococcins, treatments that provoked further lysis in addition to induced permeabilization.

Controlled permeabilization of Lactococcus lactis cells as a means to study and influence cheese ripening processes

Abstract Cells of Lactococcus lactis were permeabilized by perturbing the membrane structure through the delipidating action of n -butanol to an extent that allowed normally excluded peptidase substrates to enter the cells and be accessible to intracellular enzymes. The degree of permeabilization of cells depended on the concentration of the solvent used, the duration of the treatment, the density of the cell suspension, pH and temperature. This was indicated by the optimum or maximum activities of the intracellular peptidases aminopeptidase N and aminopeptidase X. The possible usefulness of permeabilized cells for cheese-ripening studies was demonstrated with cells that were treated with 5 ml L −1 n -butanol at pH 6.5 and 25 °C using a mixture of chymosin-generated primary cheese peptides as substrate. Production of amino acids could be correlated with conversion of peptides that had entered the permeabilized cells and reflected the course of amino acid production in cheese.

A new method for permeabilization of the plasma membrane of cultured mammalian cells. VII. Permeabilization of growing and resting cells by vortex-stirring with high molecular weight polyacrylic acid.

The permeability of murine leukemia L1210 cells toward non-permeant fluorescent Lucifer Yellow (LY) was evaluated by flow cytometry following vortex-stirring of cells with LY in the presence of high molecular weight polyacrylic acid (A-119). Permeabilization by this technique was greatly enhanced in growth phase cells, whereas it was appreciably reduced in resting cells which had been maintained in unchanged culture medium for 2 to 4d. This reduction in permeabilization of resting cells gradually disappeared when resting cells were re-suspended in fresh culture medium, and the degree of permeabilization was restored to that of the growing cells. It is recommended that the mammalian cells used for internalization of non-permeant materials by vortex-stirring with A-119 should be in the early log phase.

Studies on ribonucleoside-diphosphate reductase in permeable animal cells: I. Reversible permeabilization of mouse L cells with dextran sulfate

The treatment of mouse L cells with sodium dextran sulfate 500 for 20 min at 4 °C rendered them selectively permeable to small molecules. The degree of permeabilization was reproducible and depended on dextran sulfate concentration: as the dextran sulfate concentration increased, the cells became more permeable to the dye erythrosin B, lost the ability to incorporate [ 3H]thymidine into DNA, and acquired the capacity for nucleotide-dependent incorporation of [ 3H]dTTP into DNA and for K +- and ATP-dependent incorporation of [ 3H]lysine into protein. The ability to engage in macromolecular synthesis indicated that the permeabilized cells had retained a considerable amount of integrity, and this was confirmed by the observation that the cells could regain viability after an appropriate repair treatment. Permeabilization was rapidly reversed by incubating the cells in suspension culture medium plus 10% calf serum or in a defined medium composed of NaCl, NaH 2PO 4, glucose, glutamine, and CaCl 2, calcium ion being the most critical component. Using this minimally disruptive permeabilization procedure, it was possible to assay the enzyme ribonucleoside-diphosphate reductase in situ. The enzymatic conversion of CDP and GDP to the corresponding deoxyribonucleotides was studied; enzyme activity was almost entirely intracellular and responded to the allosteric activators and inhibitors that are known to modulate the activity of ribonucleotide reductase in vitro.