[en] Aggregation of protein is widely observed in our daily life. For example, cooking is manipulation of protein state. Main cause of various human diseases such as Alzheimer’s and Parkinson’s diseases is also considered to be aggregation of protein. One of model proteins is ovalbumin (OVA), which is a major protein in egg white. An OVA aqueous solution aggregates at high temperature and forms gel like sunny-side up above the threshold concentration. This phenomenon has been researched thoroughly from the viewpoint of turbidity, rheology, spectroscopy, scattering and so on. Then we, as chemists, think the next step for this research is manipulation of the aggregation state by modifying the chemical structure. Kawachi et al. concentrated on the N-terminal amphiphilic peptide region (pN_1_-_2_2) and proved that this peptide region enhances the strength of OVA gel from the viewpoint of rheology. In contrast, aggregation ability of OVA without this peptide region (pOVA) is dramatically reduced. We assume that the reason for this phenomenon originates from the amphiphilic nature of the peptide. The aim of this research is to clarify the role of pN_1_-_2_2 and the relationship between the microscopic chemical structure and the macroscopic physical properties. To clarify the mesoscopic structure, we conducted a SANS measurement at GP-SANS, High Flux Isotope Reactor at ORNL. Samples are solutions or gels of OVA, pOVA, peptide and their mixture with various concentrations before and after heating. pH of samples was set to 7, which is common condition for the application of OVA and their derivatives. We observed a strong upturn at low-q region in SANS curves for pOVA solutions/gels after heating. This behavior is similar to a phase separation of well-known poly(N-isopropylacrylamide) (PNIPA) solutions. From this result, we can see that the lack of amphiphilic peptide region makes the OVA solute unstable and promotes aggregation. In contrast to this, addition of amphiphilic peptide region does not change SANS profiles noticeably even after heating. This means that the peptide enhances the strength of gels without changing the original structure and is desirable for application.

[en] We have developed a surface stress sensor based on a microelectromechanical Fabry–Perot interferometer with high wavelength selectivity by using Au half-mirrors, for highly sensitive label-free biosensing. When the target molecule is adsorbed by the antigen–antibody reaction onto a movable membrane with a thin Au film, which acts as an upper mirror of the optical interferometer, the amount of deflection of the movable membrane deflected by the change in surface stress can be detected with high sensitivity. To improve the signal at the small membrane deflection region of this biosensor resulting in detection of low concentration molecules, by integrating 50 nm-thick Au half-mirrors, the wavelength selectivity of the optical interferometer has been successfully improved 6.6 times. Furthermore, the peak shift in the reflection spectrum due to the adsorption of bovine serum albumin (BSA) antigen with a concentration of 10 ng ml^−l by the antigen–antibody reaction was spectroscopically measured on the fabricated optical interferometer, and the deflection amount of the movable membrane after 10 min treatment was 2.4 times larger than that of nonspecific adsorption with the avidin molecules. This result indicated that the proposed sensor can be used for selective detection of low-concentration target antigen molecules. (paper)

No abstract available

[en] Simultaneous topography and recognition imaging (TREC) allows for the investigation of receptor distributions on natural biological surfaces under physiological conditions. Based on atomic force microscopy (AFM) in combination with a cantilever tip carrying a ligand molecule, it enables us to sense topography and recognition of receptor molecules simultaneously with nanometre accuracy. In this study we introduce optimized handling conditions and investigate the physical properties of the cantilever-tip-sample ensemble, which is essential for the interpretation of the experimental data gained from this technique. In contrast to conventional AFM methods, TREC is based on a more sophisticated feedback loop, which enables us to discriminate topographical contributions from recognition events in the AFM cantilever motion. The features of this feedback loop were investigated through a detailed analysis of the topography and recognition data obtained on a model protein system. Single avidin molecules immobilized on a mica substrate were imaged with an AFM tip functionalized with a biotinylated IgG. A simple procedure for adjusting the optimal amplitude for TREC imaging is described by exploiting the sharp localization of the TREC signal within a small range of oscillation amplitudes. This procedure can also be used for proving the specificity of the detected receptor-ligand interactions. For understanding and eliminating topographical crosstalk in the recognition images we developed a simple theoretical model, which nicely explains its origin and its dependence on the excitation frequency.

[en] Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

[en] The radioreceptorassay (RRA) has been used for measuring human chorionic gonadotropin (hCG) in sera from 751 individuals. The RRA is shown to be sensitive (98%) and specific (99.8%) in detecting hCG in a wide variety of conditions, including normal pregnancy and threatened or missed abortions. As a rapid qualitative or semiquantitative assay for hCG, the RRA is a valuable adjunct in the laboratory to less sensitive tests for hCG. Variation among different quantitative assays for hCG is examined, and it is concluded that the same assay system should be used for monitoring hCG levels in a single individual over a period of time in order to avoid inconsistent results. Application of the quantitative RRA for hCG in detecting the midcycle luteinizing hormone surge in infertillity is also presented

[en] In RNA site-directed spin labeling (SDSL) studies, structural and dynamic information at the individual RNA nucleotide level is derived from the observed electron paramagnetic resonance spectrum of a covalently attached nitroxide. A systematic approach for RNA SDSL is to establish a library that categorizes observed spectral lineshapes based on known RNA structures, thus enabling lineshape-based structure identification at any RNA site. To establish First RNA SDSL library, selective secondary structure elements have been systematically engineered into a model RNA. Nitroxide lineshapes reporting features specific to each element were obtained utilizing a new avidin-tethering scheme for suppressing spectral effects due to uniform RNA tumbling. The data demonstrated two key features required for a SDSL library with a predicting power: (i) spectral divergence-distinctive lineshape for different elements; and (ii) spectral convergence-similar lineshape for the same element in different contexts. This sets the foundation for further RNA SDSL library development

[en] Electrochemical sensing of ovalbumin (OVA) was performed using magnetic beads with OVA recognition (RNRCKGTDVQAW)/electron-transfer (YYYYC) peptides. The focus of this study was to construct a highly sensitive and regenerative tool for OVA detection based on the interaction between a protein and peptide-1(RNRCKGTDVQAWYYYYC). The peptide-1 was introduced to the bead through four types of cross-linking reagents. Magnetic beads of different sizes with N-(6-maleimidocaproyloxy)sulfosuccinimide (Sulfo-EMCS) were also prepared. An oxidation peak due to tyrosine residues at 0.65 V depended on the distance of the electron-transfer peptide from the bead surface and on the surface area of the magnetic beads that contacted the electrode surface. The response of the electro-transfer peptide moiety was decreased because the protein was accumulated via the recognition peptide on the beads. When using Sulfo-EMCS and beads that were 6.0–6.9 μm in diameter, the calibration curve of OVA was linear and ranged from 8.0 × 10"−"1"3 to 2.0 × 10"−"1"1 M. To regenerate the magnetic beads, the measurements were achieved after removal of the OVA using a denaturing reagent. When OVA was added to fetal bovine serum containing a complex matrix, OVA was recovered at a rate of 98–100%. Consequently, these magnetic beads could be a powerful tool for the sensing of OVA in real samples. - Highlights: • Ovalbumin recognition/electron-transfer peptides were immobilized on magnetic beads. • The accumulation of the protein through the peptides on the beads caused the change of electrode response. • The magnetic beads could be reused for sensing of ovalbumin.

[en] The tight junction (TJ) protein ZO-2 changes its subcellular distribution according to the state of confluency of the culture. Thus in confluent monolayers, it localizes at the TJ region whereas in sparse cultures it concentrates at the nucleus. The canine sequence of ZO-2 displays four putative nuclear export signals (NES), two at the second PDZ domain (NES-0 and NES-1) and the rest at the GK region (NES-2 and NES-3). The functionality of NES-0 and NES-3 was unknown, hence here we have explored it with a nuclear export assay, injecting into the nucleus of MDCK cells peptides corresponding to the ZO-2 NES sequences chemically coupled to ovalbumin. We show that both NES-0 and NES-3 are functional and sensitive to leptomycin B. We also demonstrate that NES-1, previously characterized as a non functional NES, is rendered capable of nuclear export upon the acquisition of a negative charge at its Ser369 residue. Experiments performed injecting at the nucleus WT and mutated ZO-2-GST fusion proteins revealed the need of both NES-0 and NES-1, and NES-2 and NES-3 for attaining an efficient nuclear exit of the respective amino and middle segments of ZO-2. Moreover, the transfection of MDCK cells with full-length ZO-2 revealed that the mutation of any of the NES present in the molecule was sufficient to induce nuclear accumulation of the protein

[en] Laser-induced breakdown spectroscopy (LIBS) was used to discern between two biological agent surrogates (Bacillus atrophaeus and ovalbumin) and potential interferent compounds (mold spores, humic acid, house dust, and Arizona road dust). Multiple linear regression and neural network analysis models were constructed by using B. atrophaeus and ovalbumin spectra, and limits of detection were calculated. Classification of the agent surrogates' LIBS spectra was attempted by using a neural network model. False negative rates of 0% were observed for B. atrophaeus (100 colony forming units) spore spectra with the neural network model used for classification