• Journal of the Korean Chemical Society
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• v.59 no.1
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• pp.22-30
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• 2015

The effect of diphtheria toxin on cell membrane lipids was studied by examining the phospholipase D (PLD) activity and free fatty acids (FFA) release in HepG2 cells. The diphtheria toxin effects on lipid alteration show apparently maximal at pH 5.1, stimulating PLD activity nearly 3.5 fold and enhancing FFA release approximately 5 fold over the control. These results indicate that the membrane is perturbed and its lipid component is rearranged during the diphtheria toxin translocation. Digitonin, a random membrane perturbing detergent, exhibit about four-fold higher perturbation effect over the diphtheria toxin at neutral pH. This observation suggests that the membrane perturbation induced by diphtheria toxin appears to be rather selective. To investigate the cause of the membrane perturbation, Cibacron blue, an inhibitor of membrane pore formation, and hemagglutinin, an influenza virus with fusion peptide, were tested for their effects on diphtheria toxin action. Cibacron blue decreased the diphtheria toxin effect by almost 50%, but the lipid alteration induced by hemagglutinin was similar to the diphtheria toxin effect. These observations imply that the membrane perturbation induced by diphtheria toxin may be caused by a combination of pore formation and insertion of hydrophobic peptide of toxin to the membrane as well. Additionally, we found that the diphtheria toxin increased the HepG2 cells permeability but the cells viability was maintained at high level at the same time. DNA fragmentation which is related to apoptosis was not induced by the toxin. Under these conditions, we could demonstrate that the lipid alteration of HepG2 cells was brought about by diphtheria toxin at acidic pH.

• Bulletin of the Korean Chemical Society
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• v.34 no.3
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• pp.931-935
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• 2013

Lipid events in liposome-mediated transfection (lipofection) are largely unknown. Here we studied whether phospholipase D (PLD), an important enzyme responsible for phospholipid breakdown, was affected during lipofection of HepG2 cells with a luciferase plasmid. Synthetic cholesterol (Chol) derivatives, including $3{\beta}$[L-ornithinamide-carbamoyl]Chol, [polyamidoamine-carbamoyl]Chol and $3{\beta}$[N-(N',N'-dimethylaminoethane)-carbamoyl]Chol, and a cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride were mixed with a helper lipid dioleoylphosphatidylethanolamine to form respective cationic liposomes. All cationic liposomes were found to stimulate PLD. Although orders of magnitude effects of the cationic liposomes on PLD stimulation did not consistently match those on cytotoxicity and luciferase expression, a causal relationship between PLD activation and cytotoxic effect was remarkable. PLD stimulation by the cationic liposomes was likely due to their amphiphilic characters, leading to membrane perturbation, as supported by similar results obtained with other membrane-perturbing chemicals such as oleate, melittin, and digitonin. Our results suggest that lipofection induces cellular lipid changes such as a PLD-driven phospholipid turnover.

• Bulletin of the Korean Chemical Society
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• v.28 no.10
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• pp.1756-1762
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• 2007

Envelope proteins of virus contain a segment of hydrophobic amino acids, called as fusion peptide, which triggers membrane fusion by insertion into membrane and perturbation of lipid bilayer structure. Potential fusion peptide sequences have been identified in the middle of L or M proteins or at the N-terminus of S protein in the envelope of human hepatitis B virus (HBV). Two 16-mer peptides representing the N-terminal fusion peptide of the S protein and the internal fusion peptide in L protein were synthesized, and their membrane disrupting activities were characterized. The internal fusion peptide in L protein showed higher activity of liposome leakage and hemolysis of human red blood cells than the N-terminal fusion peptide of S protein. Also, the membrane disrupting activity of the extracellular domain of L protein significantly increased when the internal fusion peptide region was exposed to N-terminus by the treatment of V8 protease. These results indicate that the internal fusion peptide region of L protein could activate membrane fusion when it is exposed by proteolysis.

• BMB Reports
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• v.39 no.3
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• pp.270-277
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• 2006

Helices 4 and 5 of the Bacillus thuringiensis Cry4Ba $\delta$-endotoxin have been shown to be important determinants for mosquito-larvicidal activity, likely being involved in membrane-pore formation. In this study, the Cry4Ba mutant protein containing an additional engineered tryptic cleavage site was used to produce the $\alpha4$-$\alpha5$ hairpin peptide by an efficient alternative strategy. Upon solubilization of toxin inclusions expressed in Escherichia coli and subsequent digestion with trypsin, the 130-kDa mutant protoxin was processed to protease-resistant fragments of ca. 47, 10 and 7 kDa. The 7-kDa fragment was identified as the $\alpha4$-loop-$\alpha5$ hairpin via N-terminal sequencing and mass spectrometry, and was successfully purified by size-exclusion FPLC and reversed-phase HPLC. Using circular dichroism spectroscopy, the 7-kDa peptide was found to exist predominantly as an $\alpha$-helical structure. Membrane perturbation studies by using fluorimetric calcein-release assays revealed that the 7-kDa helical hairpin is highly active against unilamellar liposomes compared with the 65-kDa activated full-length toxin. These results directly support the role of the $\alpha4$-loop-$\alpha5$ hairpin in membrane perturbation and pore formation of the full-length Cry4Ba toxin.

• Journal of the Korean Magnetic Resonance Society
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• v.11 no.2
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• pp.110-114
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• 2007

[ $^{31}P$ ] powder pattern spectra were measured to investigate the aspects of the interaction between the MLV (Multilamellar vesicle) and PMAP-23, a membrane of cathelicidin family and then CSAs(chemical shift anisotropy) were calculated to indentify the extent of perturbation of phospholipid mobility by the peptides. We found that acidic phospholipid interacts strongly with PMAP-23, and the analogues which modified to increase the amphipathic property showed that larger change of CSA. The analogue which introduced positive charge showed the same effects with amphipathic property.

• Journal of the Korean Mathematical Society
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• v.48 no.4
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• pp.867-885
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• 2011

We study a class of quasilinear wave equations with strong and nonlinear viscosity. By using the perturbation method for semilinear parabolic equations, we have established the fundamental results on existence, uniqueness and continuous dependence on data of weak solutions.

• Progress in Medical Physics
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• v.3 no.2
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• pp.43-57
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• 1992

Phase detector techique provides a unique probe to membrane recycling phenomenon by enabling dynamic monitoring of cell membrane capacitance. However, it has inherent errors due to constant changes in measurement environments. The present study analyzed several error sources to develope application criteria of this technique. and the following was found based on a theoretical analysis. The initial phase angle has to be appropriately selected to minimize the error due to perturbation of access and membrane conductances. Excitation frequency is also important to determine the initial phase angle. However. deviation of the phase angle from a predetermined initial value during the measurement period does not affect capacitance estimation to a significant degree. Despite an appropriate initial phase selection an error in scaling factor is expected for a large increase in capacitance during exocytosis. which may be overcome by iteratively correcting the scaling factor over the measurement period. These results will provide a useful guideline in practical application of this technique.

• YAKHAK HOEJI
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• v.38 no.6
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• pp.637-645
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• 1994

The effect of various drugs on the stability of the liposomal membrane of phosphatidylcholine and cholesterol was studied, employing the fluorescence self-quenching method. Calcein was entrapped into the phospholipid small unilamellar vesicles and the leakage of the fluorescence probe was monitored on adding the drug to the system. The results of the experiments showed that phenothiazine derivatives, some potent local anesthetics and surface active agents were very effective in inducing the leakage of calcein from the liposome. The leakage-inducing activity of these drug substances has been ascribed to their surface activity and the perturbation of the liposomal membrane by these substances. On the other hand drug substance with low surface activity or without amphiphilic moieties did not show any effect or only small effect on the leakage of calcein from the liposomes. The effect of lipid concentration on the stability of the liposomes was also investigated to show that the higher concentrations of lipid more drug was required to induce the leakage. The effect of surface charges of vesicles was also studied, and the results showed that the charge on the liposomes enhanced the stability of the liposomes against the leakage-inducing activity of these drug substances.

• Journal of Microbiology and Biotechnology
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• v.20 no.8
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• pp.1185-1188
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• 2010

Previously, papiliocin was isolated from the swallowtail butterfly Papilio xuthus and its antimicrobial activity was suggested. In this study, the antifungal mechanism of papiliocin against Candida albicans was investigated. Confocal laser scanning microscopy (CLSM) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence analysis indicated that papiliocin disturbed the fungal plasma membrane. Moreover, the assessment of the release of FITC-dextran (FD) from liposomes further demonstrated that the antifungal mechanism of papiliocin could have originated from the pore-forming action and that the radius of the pores was presumed to be anywhere from 2.3 to 3.3 nm.

• Journal of applied mathematics & informatics
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• v.27 no.1_2
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• pp.441-452
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• 2009

In this paper, a fifth order numerical method is presented for solving singularly perturbed differential-difference equations with negative shift. In recent papers the term negative shift has been using for delay. Similar boundary value problems are associated with expected first exit time problem of the membrane, potential in models for neuron and in variational problems in control theory. In the numerical treatment for such type of boundary value problems, first we use Taylor approximation to tackle terms containing small shifts which converts it to a boundary value problem for singularly perturbed differential equation. The two point boundary value problem is transformed into general first order ordinary differential equation system. A discrete approximation of a fifth order compact difference scheme is presented for the first order system and is solved using the boundary conditions. Several numerical examples are solved and compared with exact solution. It is observed that present method approximates the exact solution very well.