Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.     

Tomographic implementation of astronomical speckle imaging from bispectra

A bispectral method for astronomical speckle imaging utilizes an average speckle bispectrum of an object to derive its Fourier phase. There has been, however, a problem in conventional bispectral algorithm owing to difficulty in processing bispectral data in a four-dimensional (4D) space. In this paper, we propose an implementation to overcome this problem, where a one-dimensional (1D) object projection is reconstructed from a two-dimensional (2D) average bispectrum of speckle projections, and object projections so obtained at various angles are then tomographically combined into a 2D object image. In this tomographic approach, processes are separable into those for individual projection angles, implying that bispectral data required to be stored at a time are from 4D to 2D and computation time can be substantially reduced by parallelizing angle-by-angle processes. We have performed experiments using simulated and observed data, and have demonstrated the feasibility of the present approach with an achievable accuracy comparable to that of a conventional approach.     

Radiochemical study of fast fission mechanism

The reaction of²³⁸U with¹²C was studied radiochemically with the purpose of elucidating fast fission characteristics. Fast fission component was extracted in far-asymmetric mass region and interpreted as the mass diffusion following the Fokker-Planck equation. Anomalous charge dispersion widths in the corresponding mass region and a sudden increase of the whole mass distribution width at the critical energy were also observed to support the above interpretation. The reaction time of fast fission was determined to be 5·10⁻²¹ s from the width and position of the mass distribution.     

Synthesis of hyperolactones A and C

Hyperolactones A ( 1 ) and C ( 3 ) have been synthesized starting from (S)‐malic acid by a straightforward route. The unique spirolactone skeleton was efficiently constructed by one‐pot reaction as a key step. The absolute stereochemistry of hyperolactones was unambiguously established by this synthesis.     

Study of Proton Resonances in ~(22)Mg by Resonant Elastic Scattering of ~(21)Na+p and Its Astrophysical Implication in ~(18)Ne(α,p)~(21)Na Reaction Rate

Proton resonant states in ~(22)Mg have been investigated by the resonant elastic scattering of ~(21)Na+p.The ~(21)Na beam with a mean energy of 4.00 MeV/u was separated by the CNS radioactive ion beam separator(CRIB) and bombarded a thick(CH_2)_n target.The energy spectra of recoiled protons were measured at scattering angles of θ_(cm)～172° and 146°,respectively.A new state at 7.06 MeV has been observed clearly and another new one at 7.28 MeV is tentatively identified due to its low statistics.The proton resonant parameters were deduced from an R-matrix analysis of the differential cross section data.The astrophysical resonant reaction rate for the ~(18)Ne(α,p) ~(21)Na reaction has been estimated,and it is about five times larger than that assumed before.     

Magneto-optical single dot spectroscopy of GaSb/GaAs type II quantum dots

We have investigated magneto-optical properties of GaSb/GaAs self-assemble type II quantum dots by single dot spectroscopy in magnetic field. We have observed clear Zeeman splitting and diamagnetic shift of GaSb/GaAs quantum dots. The diamagnetic coefficient ranges from 5 to 30 μeV/T². The large coefficient and their large distribution are attributed to the size inhomogeneity and electron localization outside the dot. The g-factor of GaSb/GaAs quantum dots is slightly larger than that of similar type I InGaAs/GaAs quantum dots. In addition, we find almost linear relationship between the diamagnetic coefficient and the g-factor. The linear increase of g-factor with diamagnetic coefficient is due to an increase of spin-orbit interaction with dot size.     

Effects of ZnS:Mn/AlN multilayer structure on luminescent properties of nanostructured thin-film EL device

Effects of ZnS:Mn/AlN multilayer structure on luminescent properties of nanostructured (NS) thin-film electroluminescent (TFEL) device of which emission layer is a multilayer composed with ZnS:Mn layers and 0.7-nm-thick AlN interlayers were studied. The bandgap widening and the increased PL efficiency of Mn²⁺ 3d-3d transitions with a decrease in the ZnS:Mn single-layer thickness down to 5 nm were observed, which is ascribed to quantum confinement effects. Meanwhile, the multilayer with 2-nm-thick ZnS:Mn single-layers shows a drop of PL efficiency, indicating the presence of defective region just on AlN. The tendency of the luminous efficiency of the NS-TFEL device against the ZnS:Mn single-layer thickness is similar to the tendency found in the PL efficiency, indicating the importance of the ZnS:Mn/AlN interface for the device performance.     

Near Field Stimulated Time of Flight Mass Surface Analyzer

This work describes a groundbreaking process that provides a direct highly localized measurement of the atomic mass on surfaces at room temperature. Employing an original system that joins a scanning tunneling microscopy (STM) device and a time of flight (TOF) mass analyzer, we could previously ionize surface atoms by the combination of an optical laser pulse and an electric pulse at the STM tip. Desorbed ions from a localized area were accelerated and detected by a TOF chamber. We will demonstrate in this paper that high localization and mass discrimination can be obtained even without the aid of an electric pulse from the tip. We reduced the angle of incidence of the laser beam to zero (laser beam parallel to the sample surface). In this condition we were able to demonstrate for the first time ionic desorption at a confinement level of the order of 5-10nm, an order of magnitude better than previous configurations.This paper was originally presented at the 11th Optical Near Field Workshop, which was held on June 28, 2002 at Tokyo Institute of Technology, Yokohama, organized by the Optical Near Field Group of the Optical Society of Japan, an affiliate of the Japan Society of Applied Physics.