Label-free, chronological and selective detection of aggregation and fibrillization of amyloid ß protein in serum by microcantilever sensor immobilizing cholesterol-incorporated liposome.

To facilitate the early diagnosis of Alzheimer's disease and mild cognitive impairment patients, we developed a cantilever-based microsensor that immobilized liposomes of various phospholipids to detect a trace amount of amyloid ß (Aß) protein, and investigated its aggregation and fibrillization on model cell membranes in human serum. Three species of liposomes composed of different phospholipids of 1,2-dipalmtoyl-sn-glycero-3-phosphocholine (DPPC), DPPC/phosphatidyl ethanolamine and 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol having varied hydrophilic groups were applied, which showed different chronological interactions with Aß(1-40) protein and varied sensitivities of the cantilever sensor, depending on their specific electrostatic charged conditions, hydrophilicity, and membrane fluidity. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) having short hydrophobic carbon chains confirmed to show a large interaction with Aß(1-40) and a high sensitivity. Furthermore, the incorporation of cholesterol into DMPC was effective to selectively detect Aß(1-40) in human serum, which effect was also checked by quartz crystal microbalance. Finally, Aß detection of 100-pM order was expected selectively in the serum by using the developed biosensor.

A Label-Free Fluorescent Array Sensor Utilizing Liposome Encapsulating Calcein for Discriminating Target Proteins by Principal Component Analysis.

A new fluorescent arrayed biosensor has been developed to discriminate species and concentrations of target proteins by using plural different phospholipid liposome species encapsulating fluorescent molecules, utilizing differences in permeation of the fluorescent molecules through the membrane to modulate liposome-target protein interactions. This approach proposes a basically new label-free fluorescent sensor, compared with the common technique of developed fluorescent array sensors with labeling. We have confirmed a high output intensity of fluorescence emission related to characteristics of the fluorescent molecules dependent on their concentrations when they leak from inside the liposomes through the perturbed lipid membrane. After taking an array image of the fluorescence emission from the sensor using a CMOS imager, the output intensities of the fluorescence were analyzed by a principal component analysis (PCA) statistical method. It is found from PCA plots that different protein species with several concentrations were successfully discriminated by using the different lipid membranes with high cumulative contribution ratio. We also confirmed that the accuracy of the discrimination by the array sensor with a single shot is higher than that of a single sensor with multiple shots.

Classification of binding property of amyloid ß to lipid membranes: Membranomic research using quartz crystal microbalance combined with the immobilization of lipid planar membranes.

A biomembrane-related fibrillogenesis of Amyloid ß from Alzheimer' disease (Aß) is closely related to its accumulation behavior. A binding property of Aß peptides from Alzheimer' disease to lipid membranes was then classified by a quartz crystal microbalance (QCM) method combined with an immobilization technique using thiol self-assembled membrane. The accumulated amounts of Aß, Δfmax, was determined from the measurement of the maximal frequency reduction using QCM. The plots of Δfmax to Aß concentration gave the slope and saturated value of Δfmax, (Δfmax)sat that are the parameters for binding property of Aß to lipid membranes. Therefore, the Aß-binding property on lipid membranes was classified by the slope and (Δfmax)sat. The plural lipid system was described as X + Y where X = L1, L1/L2, and L1/L2/L3. The slope and (Δfmax)sat values plotted as a function of mixing ratio of Y to X was classified on a basis of the lever principle (LP). The LP violation observed in both parameters resulted from the formation of the crevice or pothole, as Aß-specific binding site, generated at the boundary between ld and lo phases. The LP violation observed only in the slope resulted from glycolipid-rich domain acting as Aß-specific binding site. Furthermore, lipid planar membranes indicating strong LP violation favored strong fibrillogenesis. Especially, lipid planar membranes indicating the LP violation only in the slope induced lateral aggregated and spherulitic fibrillar aggregates. Thus, the classification of Aß binding property on lipid membranes appeared to be related to the fibrillogenesis with a certain morphology.

Isolation and characterization of Microbulbifer species 6532A degrading seaweed thalli to single cell detritus particles.

To reduce the volume of seaweed wastes and extract polysaccharides, seaweed-degrading bacteria were isolated from drifting macroalgae harvested along the coast of Toyama Bay, Japan. Sixty-four bacterial isolates were capable of degrading "Wakame" (Undaria pinnatifida) thallus fragments into single cell detritus (SCD) particles. Amongst these, strain 6532A was the most active degrader of thallus fragments, and was capable of degrading thallus fragments to SCD particles within a day. Although the sequence similarity of the 16S rRNA gene of strain 6532A was 100% similar to that of Microbulbifer elongatus JAMB-A7, several distinct differences were observed between strains, including motility, morphology, and utilization of D: -arabinose and gelatin. Consequently, strain 6532A was classified as a new Microbulbifer strain, and was designated Microbulbifer sp. 6532A. Strain 6532A was capable of degrading both alginate and cellulose in the culture medium, zymogram analysis of which revealed the presence of multiple alginate lyases and cellulases. To the best of our knowledge, this is the first study to directly demonstrate the existence of these enzymes in Microbulbifer species. Shotgun cloning and sequencing of the alginate lyase gene in 6532A revealed a 1,074-bp open reading frame, which was designated algMsp. The reading frame encoded a PL family seven enzyme composed of 358 amino acids (38,181 Da). With a similarity of 74.2%, the deduced amino acid sequence was most similar to a Saccharophagus enzyme (alg 7C). These findings suggest that algMsp in strain 6532A is a novel alginate lyase gene.

Evaluation of Low-Temperature Sterilization using Hydrogen Peroxide Gas Containing Peracetic Acid.

In low-temperature sterilization for the medical field, hydrogen peroxide sterilization is widely used for its safety. However, its low penetrability and residual amount of sterilant are major concerns. Recently, the combination of hydrogen peroxide and peracetic acid has been found to enforce sporicidal effect, with low concentration in hydrogen peroxide. The application of this finding in medical sterilization is still very limited. To elucidate the combination effect, we compare peracetic acid containing hydrogen peroxide gas sterilizer and conventional hydrogen peroxide gas (plasma) sterilizers. The sterilant penetrability was examined in hollow load process challenge devices with inner diameters of 1 and 2 mm and lengths of 1, 2, and 3 m. As a result, peracetic acid containing hydrogen peroxide gas sterilizer demonstrated total inactivation with all diameters and lengths and achieved the highest sterilant penetrability in this study. The amount of residual sterilant on the surface of the sterilized object was 4.2 µg/cm2, which corresponds to half amount of those of conventional hydrogen peroxide gas sterilizers. These results suggest that the addition of peracetic acid to hydrogen peroxide gas sterilizer can enhance sterilization efficiency and safety.

Low temperature preparation of bismuth-related ferroelectrics powder and thin films by hydrothermal synthesis.

Bi(4)Ti(3)O(12) (BIT) thin films were prepared by low temperature hydrothermal synthesis on Pt/TiO(x)/SiO(2)/Si. Bi(4)Ti(3)O(12) or TiO(2) gel solution was formed and annealed at 350 degrees C. The BIT thin films were crystallized as a Bi-layer structural ferroelectric. During the hydrothermal treatment, the TiO(2) anatase (101) peak appears and seems to play the role as an intermediate layer. Randomly oriented BIT thin films were obtained. As a result, the BIT thin films have ferroelectric property. The as-deposited BIT thin films include spherical grains with the grain size of 120 nm.

A new SrBi4Ti4O15/CaBi4Ti4O15 thin-film capacitor for excellent electric stability.

SrBi(4)Ti(4)O(15) (SBTi) and CaBi(4)Ti(4)O(15) (CBTi) dielectric films of bismuth layered-structure dielectrics (BLSD) are prepared on Pt(100) film for constructing stacked-type dielectric capacitors; it is observed that they are c-axis singleoriented crystalline films. Compared with the perovskite barium titanate family of (Ba,Sr)TiO(3) (BST), it is observed that the SBTi film keeps a low leakage of 10(-7) A/cm(2) at 250 kV/ cm, which is smaller by an order of magnitude than the BST film, even with thinner SBTi film. The temperature coefficient of capacitance (TCC) of the SBTi or CBTi film is about 100 to 250 ppm/K and is much smaller than that of the perovskite BST film. Because the SBTi and CBTi films have opposite polarities of TCC in this experiment, they are expected to cancel out the temperature dependence in the SBTi/CBTi composite capacitor. These results indicate that the BLSD films of SBTi and CBTi are effective for application in high-temperature and high-permittivity capacitors with the practical barium perovskite oxide family.

Comparison of BST film microwave tunable devices based on (100) and (111) MgO substrates.

We have increased the figure-of-merit (FOM) of a (Ba,Sr)TiO3 (BST) film microwave tunable device by approximately three times for MgO(111) compared with a MgO(100) substrate at a frequency range of 20 GHz. Differences in permittivity and tunability in a BST film may be closely related to the difference in the film strain. The ratio of calculated permittivities of BST(100) and BST(111) films nearly corresponds to that of the FOM in the microwave range, which was rather unexpected because a higher permittivity leads to both larger tunability and dielectric loss in ferroelectrics. From a series of results, it is suggested that there are additional influences of orientation (other than the direct influence of strain itself) on the tunable properties in BST films especially in the high-frequency region.

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3.

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.