1 Department of Histology and Embryology and 2 Department of Electron Microscopy, Center of Biostructure, Medical Academy, Warsaw, Poland
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Abstract |
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Key words: cyclodextrin/embryotoxicity/hydrogen peroxide/magainine-2-amide/preimplantation mouse embryo
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Introduction |
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The mode of operation of magainin involves the formation of voltage-dependent channels leading to subsequent cell death as a consequence of ion dissipation. Magainin molecules are first adsorbed to the surface of the membrane from where they intercalate into the membrane forming ion channels (Matsuzaki et al., 1995a, 1998
). Magainins interact preferentially with membranes that are negatively charged, contain acidic phospholipids and have no cholesterol (Matsuzaki et al., 1995b
; Wieprecht et al., 1997
). These membrane attributes are characteristic of prokaryotic membranes which is why magainins are good candidates as potential antimicrobial agents.
The cell membrane of preimplantation mouse embryos presents a unique sterol composition (Pratt, 1982, 1985
) that predisposes it to magainin susceptibility.
Magainin at moderate concentration exerts a toxic effect on some tumour cell lines in vitro, melanoma cells in vivo, preimplantation mouse embryos as well as on human spermatozoa (Cruciani et al., 1991; Baker et al., 1993
; Sawicki and Mystkowska, 1999
; Wójcik et al., 2000
). At high concentrations it may affect at least some types of normal somatic cells (Chen et al., 1988
; Matsuzaki, 1998
). The efficiency in canalization of somatic cell membrane depends on the sequence of amino acids of a peptide antibiotic (Matsuzaki, 1998
). Moreover, specific modification of the extracellular environment changes the action of magainin and/or susceptibility of the cells (Chen et al., 1988
; Choi and Toyoda, 1998
; Lichtenstein et al., 1988a
; Wójcik et al., 2000
). Therefore, we tested the effects of albumin, cyclodextrin, hydrogen peroxide (H2O2) and acidification of the medium on the modification of action of magainin.
We reported previously (Sawicki and Mystkowska, 1999) that magainin is highly toxic to preimplantation mouse embryos. Therefore, the objective of the present investigation was to confirm magainin embryotoxicity towards murine 2-cell stage embryos, and also to find, and test the chemical and physical factors that enhance this toxicity. It was shown that cyclodextrin, bovine serum albumin (BSA), exogenous H2O2 and acidification of the medium (pH 6.4) enhanced the embryotoxicity of magainin when added at low (subthreshold) concentrations. The effect of magainin in vivo may presumably be enhanced by albumin, H2O2 and by acidification. Our results suggest that magainin can be used as a contraceptive molecule. Additionally, we present for the first time the effects of various concentrations of exogenous H2O2 on preimplantation mouse embryos.
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Materials and methods |
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Embryo culture and cell viability
The recovered embryos were pooled and 15 embryos transferred to a 5 µl droplet of M2 medium containing 0.4% of either polyvinyl pyrrolidone, PVP (referred to further as M2-PVP medium) or bovine serum albumin, BSA (referred to further as M2-BSA medium). In each experiment at least 7 droplets containing embryos were used. Embryos were then incubated at 37° C in a humidified atmosphere in the air, in droplets of M2 medium under paraffin oil, for 24 h. Visual assessment of cells, as well as tests of their viability, were carried out at various times during the culture.
Cell death was observed by the characteristic structural alterationsenlargement and flattening of blastomeres as well as pronounced cytoplasmic granularity. Additionally, cell death was verified by staining with 50 µg/ml of propidium iodide (Sigma) and 1.5 µmol/l fluorescein diacetate (Sigma) followed by examination under a fluorescence microscope. Propidium iodide, an exclusive vital dye, enters the cell through damaged membranes, while fluorescein diacetate is converted by nonspecific esterases to fluorescein which then penetrates into and stains living cells.
Since we observed that in 2-cell embryos, subjected to various experimental conditions, either one or both blastomeres died, our results are presented as the proportion of dead cells observed divided by the total number of cells used in the experiments.
Chemicals and treatment
Magainin-2-amide (kindly supplied by Dr W.L.Maloy, Magainin Pharmaceuticals, USA and purchased from Sigma) was dissolved in either M2-PVP or M2-BSA medium. We found in the pilot experiments that dramatic embryotoxicity appears at a minimal magainin concentration of 250 µg/ml, at which almost 100% of cells were killed within 30 min. This concentration of magainin was referred to as the threshold concentration. Therefore, in the tests of enhancement of magainin embryotoxicity we applied subthreshold concentrations of the peptide: 200 µg/ml in the tests of cyclodextrin and 166 µg/ml in the tests of H2O2 and acidification. In the subthreshold concentrations of magainin barely 2030% of cells were killed within 1224 h. In addition, the M2-BSA medium adjusted to either pH 7.4 or pH 6.4 was prepared and used for culture in medium alone (control) or with subthreshold concentration of 166 µg/ml magainin.
Methyl-ß-cyclodextrine (Sigma) was dissolved in M2-PVP medium at pH 7.4. Preincubation of embryos in 0.7 mmol/l final solution of cyclodextrin lasted for 1.5 h. The embryos were then either tested for viability or transferred to M2-PVP medium alone or to M2-PVP containing 200 µg/ml magainin (subthreshold concentration) and cultured for 24 h.
Concentrations of H2O2 from 103 mol/l to 108 mol/l were prepared by dilution of 30% H2O2 with water and then with M2-BSA medium at pH 7.4. The effect of various concentrations of H2O2 on the viability of 2-cell embryos was tested. To investigate the effect of H2O2 on the cell response to magainin the embryos were cultured in M2-BSA supplied with 107 mol/l H2O2 and a subthreshold concentration of 166 µg/ml of magainin.
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Results |
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Preincubation of 2-cell embryos for 1.5 h in M2-PVP medium containing cyclodextrin followed by the culture of cells in M2-PVP medium supplied with a subthreshold concentration of 166 µg/ml magainin, enhanced the effect of magainin. Treatment with magainin alone for 30 min or 7 h produced 5.5 and 18.5% of dead blastomeres while cells preincubated with cyclodextrin produced in the corresponding times and with the same concentrations of magainin 48 and 84% of dead blastomeres respectively (Figure 2). The blastomeres of 2-cell embryos cultured in M2-PVP medium supplied with cyclodextrin alone remained alive and showed the ability to divide in the course of 24 h (Figure 2
).
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Synergism of hydrogen peroxide and magainin
To test the enhancement of the effect of magainin on 2-cell embryos, by H2O2, we used the lowest effective concentration of 107 mol/l H2O2 and the subthreshold concentration of magainin (166 µg/ml). While both the magainin and H2O2 acting separately elicited congruent cell death rates of 5% after the 4th, 610% after the 9th and 1824% after 24 h of treatment (Figure 4), the simultaneous treatment of both substances at the same time points showed clear synergism: 36% of cells died after 1 h, 58% after 9 h and 65% after 24 h of treatment.
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Discussion |
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The cell membrane of preimplantation embryos consists of the same types of phospholipids as those of somatic cells. However, sterol synthesis starts in the 2-cell embryos and proceeds to the lanosterol step only (Pratt, 1982, 1985
). Since lanosterol makes membranes more fluid than cholesterol (Yeagle et al., 1977
; Yeagle, 1989
) the blastomere membrane of cleavage embryos could be more susceptible to magainin than that of somatic cells which contain cholesterol. The distribution of lanosterol within the blastomere membrane is noteworthy. It has a mosaic character and is highly asymmetric, being lowest at the surface of attachment to neighbouring cells (Pratt, 1982
, 1985
). This feature of blastomere membrane, together with its lanosterol content is presumably responsible for the considerable susceptibility of mouse embryos to magainin. On the other hand, the asymmetric distribution of sterols in blastomere membrane may lead to the fusion of sister blastomeres as observed here.
The present investigation indicates that both cyclodextrin and, to a lesser extent, bovine serum albumin enhanced the cytotoxic effect of magainin. While the magainin alone, at a subthreshold concentration, killed 535% of embryonic cells within 0.524 h, the supplementation of cyclodextrin, H2O2 or acidification to the medium, increased the rate of the dead cells to 85, 65 and 84% respectively. As observed previously, cyclodextrin removes sterols, including cholesterol, from cell membranes (Haynes et al., 2000). Cyclodextrin-treated spermatozoa become highly susceptible to magainin (Kilsdonk et al., 1995
; Choi and Toyoda, 1998
; Wójcik et al., 2000
). Therefore, one might speculate that the elimination of sterols from the membrane by cyclodextrin is the major cause of enhanced susceptibility of blastomeres to magainin found in this study. Like cyclodextrin, serum albumin is thought to be an extracellular acceptor of membrane sterols. However, the molecular mechanisms underlying albumin's effect on membrane cholesterol have not been clearly determined (Haynes et al., 2000
).
The minimal toxic concentration of magainin for micro- organisms was 10100 µg/ml whereas the toxicity for tumour cells was 20200 µg/ml (Zasloff, 1987). However, lysis of erythrocytes required as much as 1 mg/ml of magainin (Chen et al., 1988
; Matsuzaki et al., 1997b
).
We previously found that mouse oocytes, 1- and 2-cell embryos as well as blastocyst cells were killed in the course of a 1 h treatment with 250 µg/ml magainin (Sawicki and Mystkowska, 1999) and that this effect was dose-dependent. The action of magainin is highly sensitive to the lipid composition of the membrane (Matsuzaki, 1998
; Matsuzaki et al., 1998
). The efficiency of the binding of magainin to the membrane surface, channel size, density and stability depend on the magainin concentration. At a low concentration of magainin the proportion of peptide to membrane lipids is relatively low and the magainin molecules tend to take an orientation parallel to the lipid bilayer, thus disturbing lipid packing. However, if the proportion of peptide to membrane lipids is relatively high the magainin molecules tend to intercalate into the lipid bilayer and form transmembrane channel complexes composed of three to six molecules each (Ludtke et al., 1996
; Jo et al., 1998
; Wenk and Seelig, 1998
). Experimental models carried out using the membrane of artificial liposomes showed that the complexes are formed in the course of 30 s (Jo et al., 1998
). The prompt embryotoxic effect of magainin found in this study confirms those findings.
H2O2 is produced both inside and outside cells (Fridovich, 1998). Unbalanced production of H2O2 can induce oxidative damage to membrane lipids, proteins and DNA resulting in cellular injury either directly or indirectly through hydroperoxyradicals and lipid hydroperoxides which are precursors of the toxic end product, 4-hydroxy2nonenal (HNE) (Uchida et al., 1999
). At low concentrations exogenous H2O2 induced an adaptative response of cells by promotion of synthesis of antioxidant enzymes and heat shock proteins (Lee and Um, 1999
). H2O2 can kill cells either by apoptosis or by necrosis (Kim et al., 2000
). High doses of H2O2 can induce necrosis by impairment of the activity of respiratory enzymes, membrane channels and transporters. This may be done by selectively binding the hydroperoxyl radical to the imidazole moiety, histidine residues and sulphydryl group of cysteine residues (Uchida et al., 1999
).
For most cells protection against oxidative stress is provided by catalase, glutathione peroxidase and glutathione itself. Production of catalase is initiated in the mouse in the blastocyst stage (Harvey et al., 1995); therefore, the critical role in the protection of 2-cell embryos against oxidative stress evoked by exogenous H2O2 may be performed by hypotaurine (Guerin and Menezo, 1995
), glutathione and glutathione peroxidase. The latter is a major H2O2-consuming enzyme while the former serves as the substrate. Glutathione and its peroxidase are synthesized by oocytes and also by cumulus cells during oogenesis (de Matos et al., 1997
; El Mouatassim et al., 1999
). Cleavage mouse embryos cannot synthesize glutathione but can utilize that produced during oogenesis (Gardiner and Reed, 1995
). Extracellular glutathione has been found in preovular follicular and reproductive tract fluid (Gardiner et al., 1998
; Józwik et al., 1999
). Unfavourable embryo culture conditions raise cellular levels of H2O2 and generate the 2-cell block in mouse and rat embryos (Nasr-Esfahani et al., 1990
, Matsumoto et al., 1998
).
As indicated by the present investigation, the blastomeres of 2-cell embryos are more sensitive to exogenous H2O2 than somatic cells. For the latter cells cultivated in vitro, and for erythrocytes and leukemic cells, the LD50 was 102103mol/l (Lichtenstein et al., 1988b), while the blastomeres of 2-cell embryos died at the concentration 103105 mol/l H2O2. Our results suggest strongly that H2O2 facilitates the adsorption and binding of magainin to the lipid bilayer. This is in accordance with the fact that the oxidation of fatty acids impairs membrane integrity through elimination of polyunsaturated fatty acids and sterols (Griveau et al., 1995
) and modification of membrane charge. The results of the present investigation confirm the earlier finding (Lichtenstein et al., 1988b
) that H2O2 intensified the binding of iodinated neutrophil defensin, another type of antimicrobial peptide, to cell membrane (White et al., 1995
).
We found that culture of 2-cell embryos in medium acidified to pH 6.4 remarkably enhanced their susceptibility to magainin. The blastomeres of 2-cell embryos cultured in the medium at pH 6.4 survived well with their dividing activity deteriorating only slightly. The blastomeres of cleavage embryos can respond to the low external pH by increasing slightly their internal acidity. An excess of protons outside and within blastomere membranes is buffered by the proteins of proton channels (Baltz et al., 1991, 1993
; Dale et al., 1998
; Banfi et al., 2000
; Phillips et al., 2000
). The ability of embryos to maintain their inner/outer pH emerges at the morula/blastocyst stage when embryos encounter an acidic environment in the uterus (Dale et al., 1998
).
It is well known that the conformation and self-association of protein molecules, as well as the cytotoxicity and fusogenecity of pore-forming proteins, are pH-dependent (Kagan et al.,1992; Hu et al., 2000
). Acidification enhances the affinity of molecules to bind with the cell surface, and to insert into cell membranes. Acidified media increase the protonation of amino groups of magainin which enhances its helicity, helix stability and maximizes the lytic property of magainin (Matsuzaki et al., 1997a
). However, the mechanism of the enhancement of embryo sensitivity to magainin continues to be an enigma. It is noteworthy that the bacteria cultured in acidified medium were found to be much more efficiently permeabilized by defensin than those cultured in neutral medium (Sawyer et al., 1988
). We can, therefore, speculate that acidification can confer on molecules a conformational feature that facilitates the intercalation of magainin into both membrane and channel formation.
Both the antioxidant system of the Graffian follicle (Józwik et al., 1999) and pH-stabilization of intrafollicular fluid (Dale et al., 1998
) are efficient defence mechanisms of the preovulatory oocyte. The ovulated oocyte, zygote and preimplantation embryo are exposed to more changeable conditions in the reproductive tract. However, the physiological pH of oviductal fluid in humans is alkaline (pH 7.77.9) although in inflammation the local pH could be reduced to a value below pH 5 (Simmen and Blaser, 1993
). The sensitivity of preimplantation embryos to peroxidative damage, the devastating effect of magainin, and the synergistic effect of these factors, may be considered as the putative cause of at least some idiopathic infertilities.
The experimental systems magainin/H2O2 and magainin/acidified medium used in the present investigation may mimic, to some extent, the naturally occurring mammalian system, consisting of defensins/cellular environment at inflammation (Lichtenstein et al., 1988b). In both systems the enhancement of peptide antibiotic embryotoxicity occurs. Magainin-like immunoreactivity found in human submandibular gland (Hansen et al., 1995
) renders the potential magainin hazard more probable.
On the other hand, magainin-2-amide can present in-vivo contraceptive ability. It was recently found that 250 and 500 µg of magainin-2-amide vaginally administered exerts anti-nidatory effects in the rhesus monkey (Dhawan et al., 2000). We found that cyclodextrin and H2O2 at low concentrations (107 mol/l) clearly enhanced the embryotoxicity of magainin-2-amide. Therefore, we propose that a mixture of magainin/cyclodextrin, magainin/H2O2 or magainin/cyclodextrin/H2O2 ought to be a more potent contraceptive than magainin-2-amide alone. Furthermore, peptide antibiotics including magainin have antibacterial, antifungal and antiprotozoal activity, making them promising antimicrobial agents (Hancock, 1997
). However, if used in vivo as antibiotics, the consequences of their contraceptive side-effects must also be considered.
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Acknowledgements |
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Notes |
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References |
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Baltz, J.M., Biggers, J.D. and Lechene, C. (1991) Two-cell stage mouse embryos appear to lack mechanisms for alleviating intracellular acid loads. J. Biol. Chem., 266, 60526057.
Baltz, J.M., Biggers, J.D. and Lechene, C. (1993) A novel H+ permeability dominating intracellular pH in the early mouse embryo. Development, 118, 13531361.
Banfi, B., Maturana, A., Jaconi, S. et al. (2000) A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1. Science, 287, 138141.
Chen, H.C., Brown, J.H., Morell, J.L. and Huang, C.M. (1988) Synthetic magainin analogues with improved antimicrobial activity. FEBS Let., 236, 462466.[ISI][Medline]
Choi, Y.H. and Toyoda, Y. (1998) Cyclodextrin removes cholesterol from mouse sperm and induces capacitation in a protein-free medium. Biol. Reprod., 59, 13281333.
Cruciani, R. A., Barker, J. L., Zasloff, M. et al. (1991) Antibiotic magainins exert cytolytic activity against transformed cell lines through channel formation. Proc. Natl Acad. Sci. USA, 88, 37923796.[Abstract]
Dale, B., Ménézo, Y., Cohen, J. et al. (1998) Intracellular pH regulation in the human oocyte. Hum. Reprod., 13, 964970.[Abstract]
De Matos, D.G., Furnus, C.C. and Moses, D.F. (1997) Glutathione synthesis during in-vitro maturation of bovine oocytes: role of cumulus cells. Biol. Reprod., 57, 14201425.[Abstract]
Dhawan, L., Ghosh, D., Lalitkumar, P.G.L. et al. (2000) Anti-nidatory effect of vaginally administered (Ala8,13,18)-magainin II amide in the rhesus monkey. Contraception, 62, 3943.[ISI][Medline]
El Mouatassim, S., Guerin, P. and Menezo, Y. (1999) Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stages of maturation. Mol. Hum. Reprod., 5, 720725.
Fridovich, I. (1998) Oxygen toxicity: a radical explanation. J. Exp. Biol., 201, 12031209.
Gardiner, C.S. and Reed, D.J. (1995) Synthesis of glutathione in the preimplantation mouse embryo. Arch. Biochem. Biophys, 318, 3036.[ISI][Medline]
Gardiner, C.S., Salmen, J.J., Brandt, C.J. and Stover, S.K. (1998) Glutathione is present in reproductive tract secretions and improves development of mouse embryos after chemically induced glutathione depletion. Biol. Reprod., 59, 431436.
Griveau, J. F., Dumont, E., Renard, P. et al. (1995) Reactive oxygen species, lipid peroxidation and enzymatic defence systems in human spermatozoa. J. Reprod. Fertil., 103, 1726.[Abstract]
Guerin, P. and Ménézo, Y. (1995) Hypotaurine and taurine in gamete and embryo environement de novo synthesis via cysteine sulfinic acid pathway in oviduct cells. Zygote, 3, 333343.[ISI][Medline]
Hancock, E.W. (1997) Peptide antibiotics. Lancet, 349, 418422.[ISI][Medline]
Hansen, K.A., Kilianski, J., Newhall, K. at al. (1995) Lack of magainin-like activity in human cervical tissue. AJRI, 33, 481484.[Medline]
Harvey, M.B., Arcellana-Panlilio, M.Y., Zhang, X. et al., (1995) Expression of genes encoding antioxidant enzymes in preimplanatation mouse and cow embryos and primary bovine oviduct cultures employed for embryo coculture. Biol. Reprod., 53, 532540.[Abstract]
Haynes, M.P., Phillips, M.C. and Rothblat, G.H. (2000) Efflux of cholesterol from different cellular pools. Biochemistry, 39, 45084517.[ISI][Medline]
Hu, R.,Tang, S. and Liu, W. (2000) The pH-dependent interaction of cinnamomin with lipid membranes investigated by fluorescence methods. Biol. Chem., 381, 567573.[ISI][Medline]
Jo, E., Blazyk, J. and Boggs, J.M. (1998) Insertion of magainin into lipid bilayer detected using lipid photolabels. Biochemistry, 37, 1379113799.[ISI][Medline]
Józwik, M., WolczyDski, S., Józwik, M. and Szamatowicz, M. (1999) Oxidative stress markers in preovulatory follicular fluid in humans. Mol. Hum. Reprod., 5, 409413.
Kagan, B.L., Baldwin, R.L. and Munoz, D. (1992) Formation of ion-permeable channels by tumor necrosis factor-. Science, 255, 14271430.[ISI][Medline]
Kim, D. K., Cho, E. S. and Um, H.D. (2000) Caspase-dependent and -independent events in apoptosis induced by hydrogen peroxide. Exp. Cell, Res., 257, 8288.[ISI][Medline]
Kilsdonk, E.P., Yancey, I. G., Standt, G.W. et al. (1995) Cellular cholesterol efflux mediated by cyclodextrins. J. Biol. Chem., 270, 1725017256.
Lee, B. R. and Um, H.D. (1999) Hydrogen peroxide suppresses U937 cell death by two different mechanisms depending on its concentration. Exp. Cell, Res., 248, 430438.[ISI][Medline]
Lichtenstein, A.K., Ganz, T., Nguyen, T.M. et al. (1988a) Mechanism of target cytolysis by peptide defensins. J. Immunol., 140, 26862694.
Lichtenstein, A.K., Ganz, T., Selsted, M.E. and Lehrer, R.I. (1988b) Synergistic cytolysis mediated by hydrogen peroxide combined with peptide defensins. Cellular Immunol., 114, 104116.[ISI][Medline]
Ludtke, S.J., Heller, W.T., Harroun, T.A. et al. (1996) Membrane pores induced by magainin. Biochemistry, 35, 1372313728.[ISI][Medline]
Matsumoto, H., Shoji, N., Sugawara, S. et al. (1998) Microscopic analysis of enzyme activity, mitochondrial distribution and hydrogen peroxide in two-cell rat embryos. J. Reprod. Fertil., 113, 231238.[Abstract]
Matsuzaki, K. (1998) Magainin as paradigm for the mode of action of pore forming polypeptides. Biochim. Biophys Acta, 1376, 391400.[ISI][Medline]
Matsuzaki, K., Murase, O., Fuji, N. and Miyajima, K. (1995a) Translocation of a channel-forming antimicrobial peptide, magainin, 2, across lipid bilayers by forming a pore. Biochemistry, 34, 65216526.[ISI][Medline]
Matsuzaki, K., Sugishita, K.I., Fuji, N. and Miyajima, K. (1995b) Molecular basis for membrane selectivity of an antimicrobial peptide, magainin 2. Biochemistry, 34, 34233429.[ISI][Medline]
Matsuzaki, K., Nakamura, A., Murase, O. et al. (1997a) Modulation of magainin 2-lipid bilayer interactions by peptide charge. Biochemistry, 36, 21042111.[ISI][Medline]
Matsuzaki, K., Sugishita, K.I., Harada, M. et al. (1997b) Interactions of an antimicrobial peptide, magainin, 2, with outer and inner membranes of Gram-negative bacteria. Biochim. Biophys Acta, 1327, 119130.[ISI][Medline]
Matsuzaki, K., Sugishita, K.I., Ishibe, N. et al. (1998) Relationship of membrane curvature to the formation of pores by magainin 2. Biochemistry, 37, 1185611863.[ISI][Medline]
Nasr-Esfahani, M., Aitken, J.R. and Johnson, M.H. (1990) Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development, 109, 501507.[Abstract]
Phillips, K.P., Leveille, M.C., Claman, P. and Baltz, J.M. (2000) Intracellular pH regulation in human preimplantation embryos. Hum. Reprod., 15, 896904.
Pratt, H.P. (1982) Preimplantation mouse embryos synthesize membrane sterols. Develop. Biol.,109, 501507.
Pratt, H.P. (1985) Membrane organization in the preimplantation mouse embryo. J. Embryol. exp. Morph., 90, 101121.[ISI][Medline]
Sawicki, W. and Mystkowska, E.T. (1999) Contraceptive potential of peptide antibiotics. Lancet, 353, 464465.[ISI][Medline]
Sawyer, J.G., Martin, N.L. and Hancock, R.E. (1988) Interaction of macrophage cationic proteins with the outer membrane of Pseudomonas aeruginosa. Infect. Immun., 56, 693698.[ISI][Medline]
Simmen, H.P. and Blaser, J. (1993) Analysis of pH and pO2 in abscesses, peritoneal fluid, and drainage fluid in the presence or absence of bacterial infection during and after abdominal surgery. Am. J. Surg., 166, 2427.[ISI][Medline]
Tytler, E.M., Anantharamaiah, G.M., Walker, D.E. et al. (1995) Molecular basis for prokaryotic specificity of magainin-induced lysis. Biochemistry, 34, 43934401.[ISI][Medline]
Uchida, K., Shiraishi, M., Naito, Y. et al. (1999) Activation of stress signaling pathways by the end product of lipid peroxidation. J. Biol. Chem., 274, 22342242.
Wenk, M.R. and Seelig, J. (1998) Magainin 2 amide interaction with lipid membranes:calorimetric detection of peptide binding and pore formation. Biochemistry, 37, 39093916.[ISI][Medline]
White, S.H., Wimley, W.C. and Selsted, M.E. (1995) Structure, function, and membrane integration of defensis. Curr. Opin. Struct. Biol., 5, 521527.[ISI][Medline]
Wieprecht, T., Dathe, M., Beyermann, M. et al. (1997) Peptide hydrophobicity controls the activity and selectivity of magainin 2 amide in interaction with membranes. Biochemistry, 36, 61246132.[ISI][Medline]
Wieprecht, T., Beyermann, M. and Seelig, J. (1999) Binding of antibacterial magainin peptides to electrically neutral membranes: thermodynamics and structure. Biochemistry, 38, 1037710387.[ISI][Medline]
Wojcik, C., Sawicki, W., Marianowski, P. et al., (2000) Cyclodextrin enhances spermicidal effects of magainin-2-amide. Contraception, 62, 99103.[ISI][Medline]
Yeagle, P.L. (1989) Lipid regulation of cell membrane structure and function. FASEB J., 3, 18331842.
Yeagle, P.L., Martin, R.B., Lala, A.K. et al., (1977) Differential effects of cholesterol and lanosterol on artificial membranes. Proc. Natl Acad. Sci. USA, 74, 49244926.[Abstract]
Zasloff, M. (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc. Natl Acad. Sci. USA, 84, 54495453.[Abstract]
Submitted on December 5, 2000; accepted on April 4, 2001.