Occurrence and determination of a herbicide benthiocarb in rivers and agricultural drainages

The occurrence and determination of a herbicide, benthiocarb, 4-chlorobenzyl N,N-diethylthiolcarbamate (Saturn^®) in rivers and agricultural drainages was investigated. Benthiocarb residues were detected in water samples when it has been applied to rice paddies, after rice seedling transplantation, in concentrations of between 10.00 and 0.11 μg/l. These residues entered into rivers and agricultural drainages as a result of overflows from rice paddies. However, benthiocarb residues could not be found in water samples after the termination of its application to rice paddies, suggesting that the applied benthiocarb might be degraded by microbial and physico-chemical actions such as photochemical reactions occurring on the soil surface. Benthiocarb residue levels were higher in longer rivers than in agricultural drainage. The contribution of rainfall to the benthiocarb concentrations was shown. Benthiocarb in water was extracted with n-hexane and identified by a gas chromatography using a flame photometric detector (sulphur filter 394 nm) and a combined gas chromatography-mass spectrometry-computer system. The minimum detectable amount of benthiocarb in water sample by gas chromatography was 0.56 ng and about 100 ng of benthiocarb could be identified by a gas chromatography-mass spectrometry-computer system.     

Electron-microscopic imaging of single-walled carbon nanotubes grown on silicon and silicon oxide substrates

Direct imaging of single-walled carbon nanotubes (SWNTs) suspended on pillar-patterned Si or SiO2 substrates is investigated using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The suspended nanotubes are successfully observed by direct TEM imaging and it is seen that they have either individual or bundles of SWNTs. Low energy (< or =2 keV) SEM produces high contrast images of suspended SWNTs. On the contrary, when SWNTs contact a SiO2 substrate, they are imaged using electron-beam induced current. The image brightness depends on the length of SWNTs.     

Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant

Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expression system has been reported previously [Jiang et al., FEBS Lett., 514, 290-294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine protease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (DeltadegP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment. These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extract for the production or screening of antibodies using the cell-free expression system.     

Site-directed mutagenesis study of the antibody 2D7 which catalyzes a reaction for insertion of Cu2+ into mesoporphyrin

Monoclonal antibody 2D7 generated against a transition-state analog N-methyl mesoporphyrin catalyzes a reaction for insertion of a cupric ion into mesoporphyrin. To investigate amino acid residues responsible for the catalytic activity, site-directed mutagenesis of the amino acid residues in the third complementarity determining region of the heavy chain (CDRH3) was performed on the antigen-binding fragment (Fab) of the antibody. Recombinant Fab mutants, in which Arg95 is replaced with Ala (R95A), Asp96 with Asn (D96N) and Met97 with Gly (M97G), were examined in terms of the catalytic efficiency of the reaction (k/K(S)) and the dissociation constant for N-methyl mesoporphyrin binding (K(d)) and these values were compared with those of the wild type. The k/K(S) values of the R95A and D96N mutants were 0.96% and 1.0% of that of the wild type, respectively, whereas the M97G mutant had no detectable catalytic activity. The K(d) values of the R95A and D96N mutants were 165 and 69 times that of the wild type, respectively, while that of the M97G mutant was similar to that of the wild type. The relationship between the k/K(S) and 1/K(d) values in the wild type and the R95A and D96N mutants suggests that Arg95 and Asp96 are responsible for stabilizing the transition-state in the catalytic reaction. The results of the M97G mutant allow us to propose that Met97 plays an important role in the catalytic activity probably due to a subtle and specific conformation of the antibody.     

Shear cell development for diffusion experiments in FOTON-Satellite missions and on the ground with consideration of shear-induced convection

A shear cell technique was developed to obtain exact diffusion data. The shear cell in this study was designed for the utilization under μg-conditions, especially in the FOTON-M2 mission, but also under 1g-conditions. To minimize the influence of the shear convection, the cell size, the rotation system and the speed of the discs were optimized. To minimize free surfaces, which can cause Marangoni convection, a reservoir system providing pressure on the liquid was introduced. Using this FOTON shear cell we performed short-time diffusion experiments in the In-Sn system in a parabolic flight and under 1g conditions to investigate the influence of the shear convection quantitatively. As a result, the influence of the shear convection was so small that the mean square diffusion depth caused by the shear convection was in the order of10^{? 7}m², which is smaller than 1% of the typical value X _diff ² ≈ 10^{? 4}m² in a standard diffusion experiment using the FOTON shear cell. By using this result a correction method for the evaluation of the diffusion coefficient was established. In several ground experiments, the FOTON shear cell showed the same diffusion coefficients as from μg reference experiments within the range of errors and no obvious indication of Marangoni convection was detected. From these results we confirmed that the FOTON shear cell can be applied to μg-experiments and ground-based experiments as well.     

Self-organized inorganic nanoparticle arrays on protein lattices

Cavities formed by proteins have been utilized as the reaction chamber for the fabrication of a range of inorganic nanoparticles, providing control of the size of particles by limiting growth and preventing agglomeration. In crystal form, proteins construct molecular arrays that can provide regularly arranged sites for nanoparticles. Here we report the fabrication of nanometric iron and indium particles using ferritin, an iron-storage protein. The indium nanoparticles thus formed have uniform spherical shape with diameter of 6.6 +/- 0.5 nm, while the iron nanoparticles are somewhat irregular in shape (5.8 +/- 1.0 nm). Regular two-dimensional arrays of these nanoparticles are successfully produced by crystallizing ferritin molecules on a water-air interface using the denatured protein film method. The lattice constant of these nanoparticle arrays is 13 nm with hexagonal packing, and arrays of more than 1 microm in area can be obtained by transfer onto silicon wafer.     

Observation of HOPG by STM and contact AFM in various gas atmospheres under pressures up to 1.1 MPa

Apparatus comprising a scanning tunneling microscopy (STM) and an atomic force microscopy (AFM) has been developed for use under supra-atmospheres. Observations of highly oriented pyrolytic graphite (HOPG) were carried out by STM and contact AFM operating in air and various gas atmospheres (hydrogen, helium, neon and argon) under pressures up to 1.1 MPa. Atomic resolution images of the HOPG were obtained by STM in all the gas atmospheres studied. However, it was found that the presence of water vapor gave rise to a noise current at increased pressures. Using contact AFM, the atomic resolution in an argon atmosphere decreased with increasing pressure, while atomic images were obtained under the other gas atmospheres at 1.1 MPa.     

The nuclear actin-related protein Act3p/Arp4p is involved in the dynamics of chromatin-modulating complexes

Chromatin remodelling and histone-modifying complexes govern the modulation of chromatin structure. While components of these complexes are diverse, nuclear actin-related proteins (Arps) have been repeatedly found in these complexes from yeast to mammals. In most cases, Arps are required for functioning of the complexes, but the molecular mechanisms of nuclear Arps have as yet been largely unknown. The Arps and actin, sharing a common ancestor, are supposed to be highly similar in the three-dimensional structure of their core regions, including the ATP-binding pocket. The Arp Act3p/Arp4p of Saccharomyces cerevisiae exists within the nucleus, partly as a component of several high molecular mass complexes, including the NuA4 histone acetyltransferase (HAT) complex, and partly as uncomplexed molecules. We observed that mutations in the putative ATP-binding pocket of Act3p/Arp4p increased its concentration in the high molecular mass complexes and, conversely, that an excess of ATP or ATPgammaS led to the release of wild-type Act3p/Arp4p from the complexes. These results suggest a requirement of ATP binding by Act3p/Arp4p for its dissociation from the complexes. In accordance, a mutation in the putative ATP binding site of Act3p/Arp4p inhibited the conversion of the NuA4 complex into the smaller piccoloNuA4, which does not contain Act3p/Arp4p and exhibits HAT activity distinct from that of NuA4. Although the in vitro binding activity of ATP by recombinant Act3p/Arp4p was found to be rather weak, our observations, taken together, suggest that the ATP-binding pocket of Act3p/Arp4p is involved in the function of chromatin modulating complexes by regulating their dynamics.