Langmuir monolayer of artificial pulmonary surfactant mixtures with an amphiphilic peptide at the air/water interface: comparison of new preparations with surfacten (Surfactant TA).

Interfacial behavior was studied on the pulmonary lipid mixture containing a newly designed amphiphilic alpha-helical peptide (Hel 13-5) that consists of 13 hydrophobic and 5 hydrophilic amino acid residues. Moreover, the data obtained were compared with those of commercially available Surfacten (Surfactant TA) which has been clinically used for neonatal respiratory distress syndrome (NRDS) in Japan. Surface pressure (pi)-A and surface potential (DeltaV)-area (A) isotherms were measured for our synthetic preparations and Surfacten. Herein, a mixture of dipalmitoylphosphatidylcholine (DPPC)/egg-phosphatidylglycerol (PG)/palmitic acid (PA) (68:22:9 by weight) was used as the constituent of basic preparations. Monolayers were spread on 0.02 M Tris buffer (pH 7.4) with 0.13 M NaCl at the air/liquid interface, and the surface behavior was investigated by employing the Wilhelmy method, an ionizing electrode method, and fluorescence microscopy (FM). Cyclic compression and expansion isotherms of the prepared materials (or products) (DPPC/PG/PA/Hel 13-5) were examined to confirm the spreading and respreading ability. For the prepared products, a plateau region exists on pi-A and DeltaV-A isotherms at approximately 42 mN m(-1), indicating that Hel 13-5 is squeezed out of surface monolayers together with fluid components (PG) upon lateral compression. That is, the squeeze-out phenomenon induces a 2D-3D phase transformation. In particular, the inclination of the pi-A isotherms at X(Hel 13-5) = 0.1 in the plateau region was almost zero irrespective of the molecular area. As proposed in the earlier report (Nakahara, H.; Lee, S.; Sugihara, G.; Shibata, O. Langmuir 2006, 22, 5792-5803), an observed refluorescence phenomenon was discussed for FM measurements. This phenomenon provides evidence of the squeeze-out motion with fluid molecules. Furthermore, the cyclic pi-A and DeltaV-A isotherms show larger hysteresis areas and better respreading abilities in comparison with the previous ternary systems (DPPC/PG/Hel 13-5 and DPPC/PA/Hel 13-5) that are very important properties in pulmonary functions. FM photographs and the temperature dependence of pi-A and DeltaV-A isotherms suggest that the phase behavior of the present preparation product is very similar to that of Surfacten in terms of the domain size and in parameters such as collapse pressures, maximum DeltaV values, and so on. These results demonstrate that PG and PA even in the present preparations work well for compression-expansion cycling as is the case in the previous ternary systems, and the present preparations show comparable properties to Surfacten in vitro.

Mode of interaction of hydrophobic amphiphilic alpha-helical peptide/dipalmitoylphosphatidylcholine with phosphatidylglycerol or palmitic acid at the air-water interface.

Surface pressure (pi)-, surface potential (DeltaV)-, dipole moment (mu( perpendicular))-area (A) isotherms and morphological behavior at the air-water interface were obtained for multicomponent monolayers of two different systems for dipalmitoylphosphatidylcholine (DPPC)/egg-phosphatidylglycerol (PG) (= 68:22, by weight)/Hel 13-5 and DPPC/palmitic acid (PA) (= 90:9, by weight)/Hel 13-5 (Hel 13-5 is a newly designed 18-mer amphiphilic alpha-helical peptide with 13 hydrophobic and 5 hydrophilic amino acid residues). The phase behavior of these model systems was investigated on a subsolution of 0.02 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.4) with 0.13 M NaCl at 298.2 K by employing the Wilhelmy method, the ionizing electrode method, and fluorescence microscopy. Especially, the present study focuses on the interfacial effect of the addition of Hel 13-5 on two binary systems, DPPC/egg-PG and DPPC/PA monolayers, as the substitute for pulmonary surfactant proteins, and on the respective roles of PG and PA for the monolayers in the three-component systems. Constant kink points ( approximately 42 mN m(-1)) clearly appear on the pi-A isotherms, independent of the compositions in the ternary systems, which corresponds to the Hel 13-5 collapse pressure similar to that of SP-B and SP-C as functions in multicomponent monolayers. This implies that Hel 13-5 is squeezed out of ternary monolayers above approximately 42 mN m(-1), resulting in two- to three-dimensional phase transformation. Furthermore, Langmuir isotherms clearly show that Hel 13-5 with egg-PG is squeezed out of the DPPC/egg-PG/Hel 13-5 system, whereas only Hel 13-5 is squeezed out of the DPPC/PA/Hel 13-5 system. Cyclic compression and expansion isotherms of these systems were carried out to confirm the spreading and respreading capacities. In addition, the interfacial behavior of the ternary mixtures has been analyzed by the additivity rule. Morphological examinations and comparisons have verified the interactions of Hel 13-5 with the representative miscible mixture (DPPC/PA system) by fluorescence microscopy. Consequently, distinct morphological variations corresponding to the squeeze-out behavior are observed as a fluorescent contrast recovery. Herein, a new mechanism of the refluorescent phenomenon is proposed by varying the surface composition of Hel 13-5.

Mode of interaction of amphiphilic alpha-helical peptide with phosphatidylcholines at the air-water interface.

Surface pressure (pi)-, surface potential (deltaV)-, and dipole moment (mu(perpendicular))-area (A) isotherms and morphological behavior were examined for monolayers of a newly designed 18-mer amphiphilic alpha-helical peptide (Hel 13-5), DPPC, and DPPC/egg-PC (1:1) and their combinations by the Wilhelmy method, ionizing electrode method, fluorescence microscopy (FM), and atomic force microscopy (AFM). The newly designed Hel 13-5 showed rapid adsorption into the air-liquid interface to form interfacial films such as a SP-B function. Regardless of the composition and constituents in their multicomponent system of DPPC/egg-PC, the collapse pressure (pi(c); approximately 42 mN m(-1)) was constant, implying that Hel 13-5 with the fluid composition of egg-PC is squeezed out of Hel 13-5/DPPC/egg-PC monolayers accompanying a two- to three-dimensional phase transformation. FM showed that adding a small amount of Hel 13-5 to DPPC induced a dispersed pattern of ordered domains with a "moth-eaten" appearance, whereas shrinkage of ordered domains in size occurred for the DPPC/egg-PC mixture with Hel 13-5. Furthermore, AFM indicated that (i) the intermediate phase was formed in pure Hel 13-5 systems between monolayer states and excluded nanoparticles, (ii) protrusions necessarily located on DPPC monolayers, and (iii) beyond the collapse pressure of Hel 13-5, Hel 13-5 was squeezed out of the system into the aqueous subphase. Furthermore, hysteresis curves of these systems nicely resemble those of the DPPC/SP-B and DPPC/SP-C mixtures reported before.

A kinetic and thermodynamic study on hydrolysis of sodium laurate in aqueous phase accompanied by transfer into oil phases containing different organic additives (I).

The kinetic and thermodynamic behavior at the interface between an aqueous solution of sodium laurate (NaLA) and various oil phases comprised primarily of benzene (Bz) and/or different organic compounds including amphiphiles has been investigated in regard to the hydrolysis of NaLA accelerated at the interface, transfer of lauric acid (LA) into oil phase and reverse transfer of Bz into aqueous phase in addition to interface tension. The contact of aqueous NaLA solution with the oil phase was found to accompany the mass transfer of LA and simultaneously promote the hydrolysis of NaLA in water phase. Analysis of the change of OH- ion concentration ([OH-]) over time allowed us to treat the events as a first order reaction. From the rate constant data the activation parameters such as the activation enthalpy and entropy, both of which control the transfer of LA molecules, were determined. The parameters were found to depend greatly on varied situations of the oil phase, being clearly able to explain the physicochemical behavior of the interface. Comparing the cases where the oil phase is one of the respective single systems such as Bz, dodecane (C12) and dodecylbenzene (C12Bz), C12Bz resulted in the lowest rate constant. The transfer (or hydrolysis) rate was measured for the amphiphile-added oil systems as a function of amphiphile concentration. When 0.206 M C16OH-Bz came in contact with aqueous phase, emulsion formation at the interface layer was brought about with approximately zero activation enthalpy, leading to facile or spontaneous transfer of LA. In addition, UV absorbance representing the transfer of Bz from the oil phase to the aqueous phase also demonstrated the effects of added amphiphiles on the action of the interface.

The effect of cholesterol and monosialoganglioside (GM1) on the release and aggregation of amyloid beta-peptide from liposomes prepared from brain membrane-like lipids.

In order to investigate the influence of cholesterol (Ch) and monosialoganglioside (GM1) on the release and subsequent deposition/aggregation of amyloid beta peptide (Abeta)-(1-40) and Abeta-(1-42), we have examined Abeta peptide model membrane interactions by circular dichroism, turbidity measurements, and transmission electron microscopy (TEM). Model liposomes containing Abeta peptide and a lipid mixture composition similar to that found in the cerebral cortex membranes (CCM-lipid) have been prepared. In all, four Abeta-containing liposomes were investigated: CCM-lipid; liposomes with no GM1 (GM1-free lipid); those with no cholesterol (Ch-free lipid); liposomes with neither cholesterol nor GM1 (Ch-GM1-free lipid). In CCM liposomes, Abeta was rapidly released from membranes to form a well defined fibril structure. However, for the GM1-free lipid, Abeta was first released to yield a fibril structure about the membrane surface, then the membrane became disrupted resulting in the formation of small vesicles. In Ch-free lipid, a fibril structure with a phospholipid membrane-like shadow formed, but this differed from the well defined fibril structure seen for CCM-lipid. In Ch-GM1-free lipid, no fibril structure formed, possibly because of membrane solubilization by Abeta. The absence of fibril structure was noted at physiological extracellular pH (7.4) and also at liposomal/endosomal pH (5.5). Our results suggest a possible role for both Ch and GM1 in the membrane release of Abeta from brain lipid bilayers.

An artificially designed pore-forming protein with anti-tumor effects.

Protein engineering is an emerging area that has expanded our understanding of protein folding and laid the groundwork for the creation of unprecedented structures with unique functions. We previously designed the first native-like pore-forming protein, small globular protein (SGP). We show here that this artificially engineered protein has membrane-disrupting properties and anti-tumor activity in several cancer animal models. We propose and validate a mechanism for the selectivity of SGP toward cell membranes in tumors. SGP is the prototype for a new class of artificial proteins designed for therapeutic applications.

Nanotubules formed by highly hydrophobic amphiphilic alpha-helical peptides and natural phospholipids.

We previously reported that the 18-mer amphiphilic alpha-helical peptide, Hel 13-5, consisting of 13 hydrophobic residues and five hydrophilic amino acid residues, can induce neutral liposomes (egg yolk phosphatidylcholine) to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures induces the formation of membrane structures resembling cellular organelles such as the Golgi apparatus. In the present study we focused our attention on the effects of peptide sequence and chain length on the nanotubule formation occurring in mixture systems of Hel 13-5 and various neutral and acidic lipid species by means of turbidity measurements, dynamic light scattering measurements, and electron microscopy. We designed and synthesized two sets of Hel 13-5 related peptides: 1) Five peptides to examine the role of hydrophobic or hydrophilic residues in amphiphilic alpha-helical structures, and 2) Six peptides to examine the role of peptide length, having even number residues from 12 to 24. Conformational, solution, and morphological studies showed that the amphiphilic alpha-helical structure and the peptide chain length (especially 18 amino acid residues) are critical determinants of very long tubular structures. A mixture of alpha-helix and beta-structures determines the tubular shapes and assemblies. However, we found that the charged Lys residues comprising the hydrophilic regions of amphiphilic structures can be replaced by Arg or Glu residues without a loss of tubular structures. This suggests that the mechanism of microtubule formation does not involve the charge interaction. The immersion of the hydrophobic part of the amphiphilic peptides into liposomes initially forms elliptic-like structures due to the fusion of small liposomes, which is followed by a transformation into tubular structures of various sizes and shapes.