Formation of spherulitic amyloid ß aggregate by anionic liposomes.

Alzheimer's disease is the most common form of senile dementia. This neurodegenerative disorder is characterized by an amyloid deposition in senile plaques, composed primarily of fibrils of an aggregated peptide, amyloid ß (Aß). The modeling of a senile plaque formation on a model neuronal membrane under the physiological condition is an attractive issue. In this study, we used anionic liposomes to model the senile plaque formation by Aß. The growth behavior of amyloid Aß fibrils was directly observed, revealing that the induction of the spherulitic Aß aggregates could result from the growth of seeds in the presence of anionic liposomes. The seeds of Aß fibrils strongly interacted with negatively charged liposome and the subsequent association of the seeds were induced to form the seed cluster with many growth ends, which is advantageous for the formation of spherulitic Aß aggregates. Therefore, anionic liposomes mediated not only fibril growth but also the aggregation process. These results imply that anionic liposome membranes would affect the aggregate form of Aß fibrils. The modeling of senile plaque reported here is considered to have great potential for study on the amyloidosis.

Laser-induced propagation and destruction of amyloid beta fibrils.

The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

Permeation of a beta-heptapeptide derivative across phospholipid bilayers.

Based on a number of experiments it is concluded that the fluorescein labeled beta-heptapeptide fluoresceinyl-NH-CS-(S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the beta-heptapeptide (S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb(3+)-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled beta-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular beta-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.

Arsenic (V) induces a fluidization of algal cell and liposome membranes.

Arsenate is one of the most poisonous elements for living cells. When cells are exposed to arsenate, their life activities are immediately affected by various biochemical reactions, such as the binding of arsenic to membranes and the substitution of arsenic for phosphate or the choline head of phospholipids in the biological membranes. The effects of arsenate on the life activities of algae Chlorella vulgaris were investigated at various concentrations and exposure times. The results demonstrated that the living activities of algal cells (10(10)cells/L) were seriously affected by arsenate at a concentration of more than 7.5mg As/L within 24h. Algal cells and the artificial membranes (liposomes) were exposed to arsenate to evaluate its effects on the membrane fluidization. In the presence of arsenate, the membranes were fluidized due to the binding and substitution of arsenate groups for phosphates or the choline head on the their membrane surface. This fluidization of the biological membranes was considered to enhance the transport of toxicants across the membrane of algal cells.

Atomic force microscopy observation of highly arrayed phospholipid bilayer vesicle on a gold surface.

Tapping mode atomic force microscopy (TM-AFM) imaging of a phospholipid bilayer vesicle (liposome) immobilized on a gold surface was performed to investigate morphologies of the electrode surfaces produced through application of three different sample preparation methods. We compared both methods from a morphological viewpoint using TM-AFM images. Liposomes, composed of zwitterionic and anionic phospholipids, were prepared by extrusion. Results indicate that the surface with immobilized L1-liposome, which was fabricated by the amino coupling method, seemed to form large amounts of aggregated or fused liposomes. In contrast, L2-liposome-containing 1-octadecanthiol that was directly attached on the gold surface using thiol-gold binding force was immobilized as a uniform surface topology without liposome aggregation. Finally, we attempted to arrange individual L3-liposome, prepared by mixing zwitterionic and anionic phospholipids, onto the gold layer by electron-beam (e-beam) lithography technique. A third method, L3-liposome formation on the sensor surface, is greatly anticipated for biosensor applications.

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins.

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69+/-8% in optimal conditions. When avidin-biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously.

Liposome-assisted activity of superoxide dismutase under oxidative stress.

A biological membrane is the front line of defense for cells against various environmental stresses such as heat and reactive oxygen species (ROS) and is expected to play an important role in the antioxidant system with antioxidant enzymes, similarly to its chaperone-like function in cooperation with heat shock proteins. The oxidative stress response of superoxide dismutase (SOD), which is known to catalyze the dismutation of O(2)(-) to H(2)O(2), was investigated in the presence of artificial membranes, liposomes, in order to obtain fundamental information on the biological ROS scavenging system. SOD lost its activity in the presence of H(2)O(2) and was found to have two loops including one which contains an alpha-helix which presents the substrate O(2)(-) to the activity center of SOD (Cu(II)). From circular dichroism analysis of SOD in the presence of H(2)O(2), the contents of the alpha-helix in SOD were found to decrease in correspondence with the inactivation and conformational change of SOD, suggesting that the conformation of the alpha-helix loops affects SOD activity. In the presence of liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), SOD was not inactivated in the presence of H(2)O(2) although the contents of its alpha-helix structure were decreased. The oxidized SOD was found to interact with the liposome surface under oxidative stress using dielectric dispersion analysis. Based on these results, a possible mechanism of SOD protection against ROS on liposomes was presented.

Oxidation of cholesterol catalyzed by amyloid beta-peptide (Abeta)-Cu complex on lipid membrane.

A catalytic reaction of H2O2 production by an amyloid beta-peptide (Abeta)-Cu complex with cholesterol incorporated in a liposome was kinetically analyzed. The Michaelis-Menten model was applied to the H2O2 production reaction using cholesterol as the substrate catalyzed by the Abeta-Cu complex. The Km value for the Abeta-Cu complex catalytic reaction with cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes (Km=0.436 microM for Abeta(1-40); Km=0.641 microM for Abeta(1-42)) was found to be smaller than that with cholesterol-containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes (Km=0.585 microM for Abeta(1-40), Km=0.890 microM for Abeta(1-42)). The results imply that membrane properties could play an important role in the interactions of the Abeta-Cu complex with cholesterol in these liposomes. Considering the physical states of the cholesterol/POPC (liquid disordered phase) and cholesterol/DPPC (liquid ordered phase) liposomes in the present reaction conditions, the data obtained suggests that the H2O2-generating activity of the Abeta-Cu complex, accompanied by oxidation of membrane-incorporated cholesterol, could be effected by the phase of the liposome membranes.

Heat-enhanced production of chitosanase from Streptomyces griseus in the presence of liposome.

The effects of heat stress and liposome treatment on the growth of Streptomyces griseus cells and chitosanase production were investigated on the basis of using the designed strategy of a stress-mediated bioprocess. The effective conditions for increasing the interaction between chitosanase and the 1-palmitoyl-2-oleoyl-3-phosphocholine (POPC) liposome under heat stress condition were determined on the basis of the results of circular dichroism (CD) and dielectric dispersion analysis (DDA). Under these effective conditions, S. griseus cells were cultivated for the effective production of chitosanase. The results obtained from both CD spectra and DDA showed that heat stress enhances the interaction of the POPC liposomes and chitosanase. The strongest interaction between them could be obtained in the specific temperature range of 40-45 degrees C. The enhancement of the target chitosanase production was conducted under heat stress at 41 degrees C in the presence and absence of the POPC liposomes. The growth rates of S. griseus cells in the cases of heat (41 degrees C) and heat (41 degrees C)/POPC treatments were respectively 1.2 and 1.4 times higher than that obtained under the control condition. In the heat (41 degrees C) and heat (41 degrees C)/POPC treatments, chitosanase activity increased to 1.8 and 2 times, respectively, higher than that obtained under the control condition. Heat stress and the addition of the POPC liposomes could therefore be utilized to induce the potential functions of bacterial cells for the enhancement of the final target production.

Detection of a heat stress-mediated interaction between protein and phospholipid membrane using dielectric measurement.

The dielectric response of lipid bilayer membrane vesicles (liposomes) prepared using either phosphatidylcholine from egg (EPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was analyzed at a frequency range of 0.1 to 100 MHz. A marked dielectric dispersion for EPC and POPC liposome suspensions was observed above 1 MHz. An appropriate analysis of the dielectric dispersion curve was performed using the Cole-Cole equation and the Debye equation and was found to provide a method for the determination of dielectric parameters. Among the dielectric parameters, the characteristic frequency of a second dispersion around 50 MHz varied corresponding with changes in the test conditions. Of particular note is that an anomalous change in the characteristic frequency in the presence of protein corresponded to the degree of hydrophobic interaction between proteins and liposomes. The value of the frequency around 50 MHz, as well as the decrease in permittivity over the frequency range tested, are indicators of the interaction between proteins and liposomes.

Growth behavior of giant vesicles using the electroformation method: effect of proteins on swelling and deformation.

The growth of giant vesicles (GVs) can be considered as a consecutive process of swelling/detachment/deformation, which is a response of lipid membranes on solid surfaces to the solvent and environmental factors such as temperature and ionic strength. The electroformation method allows to visualize the responses to such factors. The additive effect of the protein on the growth of GVs, composed of zwitterionic phospholipids, was herein investigated using the electroformation method. Proteins denatured by a pH-shift (to be in the Molten Globule state) perturbed the lipid membranes, resulting in the acceleration of GV growth. The GVs detached from the electrode showed deformation close to a stomatocyte. It was revealed that common factor for the response of lipid membranes was the variation of the apparent area elastic modulus associated with the interaction between proteins and lipid membranes. The present finding affords better understanding about the response of lipid membranes on solid surfaces under a variety of environment factors.

Oligolamellar vesicles for covalent immobilization and stabilization of D-amino acid oxidase.

Oligolamellar phospholipid vesicles incorporated with d-amino acid oxidase from porcine kidney (OV-DAO) were prepared by encapsulating pre-formed enzyme-bound unilamellar vesicles (UV-DAO) with bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The bilayer of UV-DAO was composed of POPC, 30 mol% of cholesterol and 15 mol% of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (NGPE) that was responsible for covalent linking to D-amino acid oxidase (DAO). OV-DAO and UV-DAO showed the activity to catalyze the oxidation of D-alanine as measured based on the hydrogen peroxide produced. The oligolamellar and unilamellar structure of OV-DAO and UV-DAO, respectively was elucidated based on the quenching characteristics of bilayers-incorporated fluorescent lipid 7-nitro-2,1,3-benzoxadiazol-4-yl-phosphoethanolamine (NBD-PE) and the size distribution of the vesicles measured with the dynamic light scattering method. The enzyme activity of OV-DAO and UV-DAO was significantly stabilized at 50°C compared to that of free DAO at the fixed enzyme concentration of 3.29 µg/mL. At the temperature, OV-DAO and UV-DAO showed the remaining activity of 52.7 and 29.6%, respectively at the incubation time of 20 min while free DAO was completely deactivated. Thus the dimeric form of DAO could be stabilized by its coupling to the surface of UV-DAO membrane being the inner bilayer of OV-DAO. Furthermore, the thermal denaturation of DAO and dissociation of flavin adenine dinucleotide (FAD) from the subunits of enzyme were prevented in the aqueous phase formed between the bilayers of OV-DAO.

Membrane fusion mediated by phospholipase C under endosomal pH conditions.

Phospholipase C (PLC) is considered to be one of key enzymes for the design of drug delivery system using the endocytosis route, because PLC can catalyze the membrane fusion between cell membranes and phospholipid vehicles (liposomes). Membrane fusion by PLC was then studied under various pHs to model the endosomal environment. The used liposomes were composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Ch). The membrane fusion was dominated by the enzymatic reaction at pH 6-7.5. In contrast, the membrane perturbation effect due to the conformational change of PLC could induce the membrane fusion at around pH 4. The maximal value of membrane fusion was observed at around pH 5 for three liposomes in the order of DOPC

Oxidative stress can affect the gene silencing effect of DOTAP liposome in an in vitro translation system.

Oxidative stress can affect in vitro GFP expression through its control of the gene silencing effect of the liposome prepared by 1,2-dioleoyl-3-trimethyl-ammonium propane (DOTAP). The gene silencing effect of cationic DOTAP liposome in in vitro GFP expression, especially focusing on its translation process, and the effects of oxidative stress on its silencing effect were investigated. GFP expression, initiated by mRNA, was found to be thoroughly inhibited in the presence of DOTAP liposome at concentration of more than 2.5 mM, though its inhibitory effect was reduced in the presence of hydrogen peroxide. The analyses of (i) the interaction of mRNA with DOTAP, (ii) the chemical structure of DOTAP, and (iii) the membrane fluidity of DOTAP liposome imply the possible role of gene expression by the liposome membrane and stress conditions.

Copper-mediated growth of amyloid ß fibrils in the presence of oxidized and negatively charged liposomes.

Amyloid ß protein (Aß) from Alzheimer's disease formed fibrillar aggregates and their morphology depended on oxidized and negatively charged liposomes. The morphology of fibrillar aggregates was affected by Cu(2+), together with their growth kinetics. This is because Cu(2+) inhibited the nucleation step in the formation of amyloid Aß fibrillar aggregates by forming Aß/Cu complex inactive to the growth of fibrillar aggregates. In addition, this is probably because Cu(2+) affected the fibrillar aggregate formed on the surface of liposomes. These findings would give a better understanding of the formation mechanism of amyloid fibrils on biomembranes.

Relationship between the mobility of phosphocholine headgroups of liposomes and the hydrophobicity at the membrane interface: a characterization with spectrophotometric measurements.

In this study, we investigated the dynamics of a membrane interface of liposomes prepared by eight zwitterionic phosphatidylcholines in terms of their headgroup mobility, with spectroscopic methods such as dielectric dispersion analysis (DDA), fluorescence spectroscopy. The DDA measurement is based on the response of the permanent dipole moment to a driving electric field and could give the information on the axial rotational Brownian motion of a headgroup with the permanent dipole moment. This motion depended on kinds of phospholipids, the diameter of the liposomes, and the temperature. The activation energy required to overcome the intermolecular force between headgroups of phospholipids depended on the strength of the interaction between headgroups such as hydrogen bonds and/or dipole-dipole interaction. Hydration at the phosphorous group of phospholipid and the molecular order of lipid membrane impaired the interaction between headgroups. Furthermore, the hydrophobicity of membrane surface increased parallel to the increase in headgroup mobility. It is, therefore, concluded that hydration of headgroup promoted its mobility to make the membrane surface hydrophobic. The lipid membrane in liquid crystalline phase or the lipid membrane with the larger curvature was more hydrophobic.

Chitosanase displayed on liposome can increase its activity and stability.

The strategy to prepare a novel biocatalyst by the immobilization of chitosanase onto liposome (ICL) was carried out based on the direct interaction of liposomes with cell membrane of Streptomyces griseus cell. The ICL was characterized in relation to the molecular weight of protein, the chitosanase activity, the effect of the surface hydration of various liposomes on hydrolysis activity of immobilized chitosanase and the stability of ICL under various extreme conditions. The SDS-PAGE analysis of the purified ICL sample shows the existence of a protein with approximately 39kDa that corresponded to the sum of weight of the mature chitosanase and its signal peptide (38.8kDa). The above protein of ICL also expresses the chitosanase activity that is significantly higher than that of the conventional chitosanase. Furthermore, the surface hydration of liposomes used to prepare ICL that affected the activity of immobilized chitosanase verified the importance of liposome surfaces. Indeed, the stability of ICL assayed by measuring the chitosanase activity is significantly higher than that of conventional chitosanase under various temperatures and pH conditions. These characteristics of ICL show the possible preparation of the biocatalysts that can be prepared by immobilizing enzymes onto liposome vesicles properly.

Analysis of the 22-NBD-cholesterol transfer between liposome membranes and its relation to the intermembrane exchange of 25-hydroxycholesterol.

The transfer of 22-NBD-cholesterol (22-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol) between two liposome membranes was quantitatively analyzed by using the fluorescence resonance energy transfer (FRET) method. Liposomes labeled with both 22-NBD-cholesterol and a rhodamine-labeled phosphatidylethanolamine (Rh-DHPE) were used as donor liposomes, and the 22-NBD-cholesterol transfer from these donor liposomes to acceptor liposomes prepared from same type of phosphatidylcholine was monitored. The transfer kinetics was found to be composed of a fast and a slow phase, and all kinetic measurements could be fitted with a bi-exponential model. The results obtained indicate that the 22-NBD-cholesterol transfer kinetics between liposome membranes depends on the fluidity of the liposome used and that the curvature may affect the kinetics. Furthermore, the behavior of 22-NBD-cholesterol in lipid membrane is similar to that of the oxysterol 25-hydroxycholesterol rather than cholesterol. It is proposed that 22-NBD-cholesterol can be a useful fluorescent probe to mimic the intermembrane transfer of oxidized cholesterols like 25-hydroxycholesterol, rather than that of cholesterol itself.

Development of liposome-based mimics of superoxide dismutase and peroxidase based on the "LIPOzyme" concept.

An antioxidative liposome catalyst, LIPOzyme, that mimics both superoxide dismutase (SOD) and peroxidase (POD)-like activities has been developed by using liposomes modified with simple ligands (dodecanoyl-histidine, Dodec-His) and metal ions (Mn). The SOD-like activity is dependent on the stability of the ligand-metal complex on the liposome membrane, with the value being higher for the DPPC liposome and at a higher pH. The POD-like activity was found to be maximal in the case of DMPC liposome, in which the ligand-metal complex is inserted more deeply into the membrane. It was thus shown that liposome modified with simple ligands can exhibit different enzyme-like activities depending on the characteristics of the liposome membrane.