Quantitative Measurements of Intercellular Adhesion Strengths between Cancer Cells with Different Malignancies Using Atomic Force Microscopy.

Intercellular adhesion strengths between two kinds of murine breast cancer cells with different malignancies were measured quantitatively using a metal cup-attached chip with atomic force microscopy (AFM). The cup-attached chip was used to approach a cell, pick it up, and then approach another cell, and the adhesion strengths were measured according to the contact time of the cells between 0 to 60 s. Separation work was used as a parameter for quantitative comparisons of the strengths. As a result, the work of a highly metastatic cancer cell (FP10SC2) was greater than a low metastatic cancer cell (4T1-LM) throughout all contact times examined. Adhesion was analyzed from a point of a view of binding kinetics of receptors on cells, and two possibilities were found: one was the number of cell adhesive receptors increased, and the other was the work to separate single molecular binding increased with increasing cancer cell malignancy. These results indicated quantitative measurements of intercellular adhesion strengths using AFM yielded information to understand the mechanism of the cancer progression from a new perspective.

Structure and orientation of bovine lactoferrampin in the mimetic bacterial membrane as revealed by solid-state NMR and molecular dynamics simulation.

Bovine lactoferrampin (LFampinB) is a newly discovered antimicrobial peptide found in the N1-domain of bovine lactoferrin (268-284), and consists of 17 amino-acid residues. It is important to determine the orientation and structure of LFampinB in bacterial membranes to reveal the antimicrobial mechanism. We therefore performed (13)C and (31)P NMR, (13)C-(31)P rotational echo double resonance (REDOR), potassium ion-selective electrode, and quartz-crystal microbalance measurements for LFampinB with mimetic bacterial membrane and molecular-dynamics simulation in acidic membrane. (31)P NMR results indicated that LFampinB caused a defect in mimetic bacterial membranes. Ion-selective electrode measurements showed that ion leakage occurred for the mimetic bacterial membrane containing cardiolipin. Quartz-crystal microbalance measurements revealed that LFampinB had greater affinity to acidic phospholipids than that to neutral phospholipids. (13)C DD-MAS and static NMR spectra showed that LFampinB formed an α-helix in the N-terminus region and tilted 45° to the bilayer normal. REDOR dephasing patterns between carbonyl carbon nucleus in LFampinB and phosphorus nuclei in lipid phosphate groups were measured by (13)C-(31)P REDOR and the results revealed that LFampinB is located in the interfacial region of the membrane. Molecular-dynamics simulation showed the tilt angle to be 42° and the rotation angle to be 92.5° for Leu(3), which are in excellent agreement with the experimental values.

Inhibitory mechanism of pancreatic amyloid fibril formation: formation of the complex between tea catechins and the fragment of residues 22-27.

Islet amyloid polypeptide (IAPP) is a major component of pancreatic amyloid deposits associated with type 2 diabetes. Polyphenols contained in plant foods have been found to inhibit amyloid fibril formation of proteins and/or peptides. However, the inhibition mechanism is not clear for a variety of systems. Here the inhibition mechanism of green tea polyphenols, catechins, on amyloid fibril formation of the IAPP fragment (IAPP22-27), which is of sufficient length for formation of ß-sheet-containing amyloid fibrils, was investigated by means of kinetic analysis. A quartz crystal microbalance (QCM) determined that the association constants of gallate-type catechins [epicatechin 3-gallate (ECg) and epigallocatechin 3-gallate] for binding to IAPP22-27 immobilized on the gold plate in QCM were 1 order of magnitude larger than those of the free IAPP22-27 peptide, and also those of epicatechin and epigallocatechin. Kinetic analysis using a two-step autocatalytic reaction mechanism revealed that ECg significantly reduced the rate constants of the first nucleation step of amyloid fibril formation, while the rate of autocatalytic growth was less retarded. (1)H nuclear magnetic resonance studies clarified that a IAPP22-27/ECg complex clearly forms as viewed from the (1)H chemical shift changes and line broadening. Our study suggests that tea catechins specifically inhibit the early stages of amyloid fibril formation to form amyloid nuclei by interacting with the unstructured peptide and that this inhibition mechanism is of great therapeutic value because stabilization of the native state could delay the pathogenesis of amyloid diseases and also the toxicity of the small oligomer (protofibril) is reported to be greater than that of the mature fibril.

Suppressed or recovered intensities analysis in site-directed (13)C NMR: assessment of low-frequency fluctuations in bacteriorhodopsin and D85N mutants revisited.

The first proton transfer of bacteriorhodopsin (bR) occurs from the protonated Schiff base to the anionic Asp 85 at the central part of the protein in the L to M states. Low-frequency dynamics accompanied by this process can be revealed by suppressed or recovered intensities (SRI) analysis of site-directed (13)C solid-state NMR spectra of 2D crystalline preparations. First of all, we examined a relationship of fluctuation frequencies available from [1-(13)C]Val- and [3-(13)C]Ala-labeled preparations, by taking the effective correlation time of internal methyl rotations into account. We analyzed the SRI data of [1-(13)C]Val-labeled wild-type bR and D85N mutants, as a function of temperature and pH, respectively, based on so-far assigned peaks including newly assigned or revised ones. Global conformational change of the protein backbone, caused by neutralization of the anionic D85 by D85N, can be visualized by characteristic displacement of peaks due to the conformation-dependent (13)C chemical shifts. Concomitant dynamics changes if any, with fluctuation frequencies in the order of 10(4) Hz, were evaluated by the decreased peak intensities in the B-C and D-E loops of D85N mutant. The resulting fluctuation frequencies, owing to subsequent, accelerated dynamics changes in the M-like state by deprotonation of the Schiff base at alkaline pH, were successfully evaluated based on the SRI plots as a function of pH, which were varied depending upon the extent of interference of induced fluctuation frequency with frequency of magic angle spinning or escape from such interference. Distinguishing fluctuation frequencies between the higher and lower than 10(4) Hz is now possible, instead of a simple description of the data around 10(4) Hz available from one-point data analysis previously reported.

Induction of cell-cell connections by using in situ laser lithography on a perfluoroalkyl-coated cultivation platform.

This article describes a novel laser-directed microfabrication method carried out in aqueous solution for the organization of cell networks on a platform. A femtosecond (fs) laser was applied to a platform culturing PC12, HeLa, or normal human astrocyte (NHA) cells to manipulate them and to facilitate mutual connections. By applying an fs-laser-induced impulsive force, cells were detached from their original location on the plate, and translocated onto microfabricated cell-adhesive domains that were surrounded with a cell-repellent perfluoroalkyl (R(f)) polymer. Then the fs-laser pulse-train was applied to the R(f) polymer surface to modify the cell-repellent surface, and to make cell-adhesive channels of several µm in width between each cell-adhesive domain. PC12 cells elongated along the channels and made contact with others cells. HeLa and NHA cells also migrated along the channels and connected to the other cells. Surface analysis by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) confirmed that the R(f) polymer was partially decomposed. The method presented here could contribute not only to the study of developing networks of neuronal, glial, and capillary cells, but also to the quantitative analysis of nerve function.

Interaction of tea catechins with lipid bilayers investigated by a quartz-crystal microbalance analysis.

The quartz-crystal microbalance (QCM) technique was applied to investigate the interaction of tea catechins with lipid bilayers. The association constants obtained from the frequency changes of QCM revealed that (-)epicatechin gallate and (-)epigallocatechin gallate interacted with 1,2-dimyristoyl-sn-glycero-3-phosphocholine ca. 1000 times more strongly than (-)epicatechin and (-)epigallocatechin. The results exhibited good correlation with the strength of biological activity.

Interactions of bovine lactoferricin with acidic phospholipid bilayers and its antimicrobial activity as studied by solid-state NMR.

Bovine lactoferricin (LfcinB) is an antimicrobial peptide released by pepsin cleavage of lactoferrin. In this work, the interaction between LfcinB and acidic phospholipid bilayers with the weight percentage of 65% dimyristoylphosphatidylglycerol (DMPG), 10% cardiolipin (CL) and 25% dimyristoylphosphatidylcholine (DMPC) was investigated as a mimic of cell membrane of Staphylococcus aureus by means of quartz crystal microbalance (QCM) and solid-state (31)P and (1)H NMR spectroscopy. Moreover, we elucidated a molecular mechanism of the antimicrobial activity of LfcinB by means of potassium ion selective electrode (ISE). It turned out that affinity of LfcinB for acidic phospholipid bilayers was higher than that for neutral phospholipid bilayers. It was also revealed that the association constant of LfcinB was larger than that of lactoferrin as a result of QCM measurements. (31)P DD-static NMR spectra indicated that LfcinB interacted with acidic phospholipid bilayers and bilayer defects were observed in the bilayer systems because isotropic peaks were clearly appeared. Gel-to-liquid crystalline phase transition temperatures (Tc) in the mixed bilayer systems were determined by measuring the temperature variation of relative intensities of acyl chains in (1)H MAS NMR spectra. Tc values of the acidic phospholipid and LfcinB-acidic phospholipid bilayer systems were 21.5 degrees C and 24.0 degrees C, respectively. To characterize the bilayer defects, potassium ion permeation across the membrane was observed by ISE measurements. The experimental results suggest that LfcinB caused pores in the acidic phospholipid bilayers. Because these pores lead the permeability across the membrane, the molecular mechanism of the antimicrobial activity could be attributed to the pore formation in the bacterial membrane induced by LfcinB.

Advanced LSI-based amperometric sensor array with light-shielding structure for effective removal of photocurrent and mode selectable function for individual operation of 400 electrodes.

We have developed a large-scale integrated (LSI) complementary metal-oxide semiconductor (CMOS)-based amperometric sensor array system called "Bio-LSI" as a platform for electrochemical bio-imaging and multi-point biosensing with 400 measurement points. In this study, we newly developed a Bio-LSI chip with a light-shield structure and a mode-selectable function with the aim of extending the application range of Bio-LSI. The light shield created by the top metal layer of the LSI chip significantly reduces the noise generated by the photocurrent, whose value is less than 1% of the previous Bio-LSI without the light shield. The mode-selectable function enables the individual operation of 400 electrodes in off, electrometer, V1, and V2 mode. The off-mode cuts the electrode from the electric circuit. The electrometer-mode reads out the electrode potential. The V1-mode and the V2-mode set the selected sensor electrode at two different independent voltages and read out the current. We demonstrated the usefulness of the mode-selectable function. First, we displayed a dot picture based on the redox reactions of 2.0 mM ferrocenemethanol at 400 electrodes by applying two different independent voltages using the V1 and V2 modes. Second, we carried out a simultaneous detection of O2 and H2O2 using the V1 and V2 modes. Third, we used the off and V1 modes for the modification of the osmium-polyvinylpyridine gel polymer containing horseradish peroxidase (Os-HRP) at the selected electrodes, which act as sensors for H2O2. These results confirm that the advanced version of Bio-LSI is a promising tool that can be applied to a wide range of analytical fields.

Interaction of extracellular loop II of κ-opioid receptor (196-228) with opioid peptide dynorphin in membrane environments as revealed by solid state nuclear magnetic resonance, quartz crystal microbalance and molecular dynamics simulation.

κ-Opioid receptor is a member of the opioid receptor family and selectively interacts with the opioid peptide dynorphin. Extracellular loop II (ECL-II) of the κ-opioid receptor displays an amphiphilic helix in membrane environments and the N-terminal α-helix of dynorphin A(1-17) (hereafter DynA17) is inserted into the membrane with the tilt angle of 21° to the bilayer normal. ECL-II peptides (1-33), corresponding to 196-228 of κ-opioid receptor with [1-(13)C]- or [3-(13)C]-labeled amino acids were incorporated into large [dimyristoylphosphatidyl choline (DMPC)/ dihexanoylphosphatidyl choline (DHPC) = 3, q = 3] and small bicelle (q = 1) systems. (13)C direct detection with dipolar decoupling and magic angle spinning (DD-MAS) nuclear magnetic resonance (NMR) spectra were recorded, and the (13)C chemical shift perturbation clearly indicated that DynA17 interacts with ECL-II at the location of Val10-Ala15. Quartz crystal microbalance measurements were performed to determine the binding constant of ECL-II with DynA17 and indicated that the binding constant between DynA17 and ECL-II embedded in the lipid layer was 72 times larger than that between DynA17 and the lipid. The result of the molecular dynamics simulation clearly indicates that the C-terminus of DynA17 interact with the amino acid residues of the region between Val10-Gln14 of ECL-II. These results suggest that DynA17 interacts with the ECL-II of the κ-opioid receptor through a hydrophobic and short-lived electrostatic interaction with high affinity in the outer surface of the membrane.

Contribution of nanoscale curvature to number density of immobilized DNA on gold nanoparticles.

We report the curvature size dependence of the density of attached single-stranded DNA (ssDNA) on the surface of gold nanoparticles. The densities of immobilized ssDNA on 10, 20, 30, and 50 nm gold nanoparticles were examined, and we found that the maximum density of the immobilized ssDNA on 10 nm particles was 13 times larger than that on 50 nm particles, which was still 10 times larger than that on flat gold surfaces. This result indicates the importance of curvature in the nanometer-scale attachment of ssDNAs to nanoparticles.

Significance of low-frequency local fluctuation motions in the transmembrane B and C alpha-helices of bacteriorhodopsin, to facilitate efficient proton uptake from the cytoplasmic surface, as revealed by site-directed solid-state 13C NMR.

13C NMR spectra of [1-13C]Val- or -Pro-labeled bacteriorhodopsin (bR) and its single or double mutants, including D85N, were recorded at various pH values to reveal conformation and dynamics changes in the transmembrane alpha-helices, in relation to proton release and uptake between bR and the M-like state caused by modified charged states at Asp85 and the Schiff base (SB). It was found that the D85N mutant acquired local fluctuation motion with a frequency of 10(4) Hz in the transmembrane B alpha-helix, concomitant with deprotonation of SB in the M-like state at pH 10, as manifested from a suppressed 13C NMR signal of the [1-13C]-labeled Val49 residue. Nevertheless, local dynamics at Pro50 neighboring with Val49 turned out to be unchanged, irrespective of the charged state of SB as viewed from the 13C NMR of [1-13C]-labeled Pro50. This means that the transmembrane B alpha-helix is able to acquire the fluctuation motion with a frequency of 10(4) Hz beyond the kink at Pro50 in the cytoplasmic side. Concomitantly, fluctuation motion at the C helix with frequency in the order of 10(4) Hz was found to be prominent, due to deprotonation of SB at pH 10, as viewed from the 13C NMR signal of Pro91. Accordingly, we have proposed here a novel mechanism as to proton uptake and transport based on a dynamic aspect that a transient environmental change from a hydrophobic to hydrophilic nature at Asp96 and SB is responsible for the reduced p Ka value which makes proton uptake efficient, as a result of acquisition of the fluctuation motion at the cytoplasmic side of the transmembrane B and C alpha-helices in the M-like state. Further, it is demonstrated that the presence of a van der Waals contact of Val49 with Lys216 at the SB is essential to trigger this sort of dynamic change, as revealed from the 13C NMR data of the D85N/V49A mutant.

Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate.

The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function.