Susumu Yoshikawa

Phenotypic diversity of Pseudoalteromonas citrea from different marine habitats and emendation of the description

Four strains of marine, aerobic, agar-decomposing bacteria with one polar flagellum and with DNA G + C contents of 38.9-40.2 mol% were isolated from the Far-Eastern mussels Crenomytilus grayanus and Patinopecten yessoensis. These four strains were identified as Pseudoalteromonas; however, they were phenotypically different from species described previously according to carbon compound utilization tests and the BIOLOG identification system. High agar-decomposing activity was found in two strains, in one of which agarase, alpha-galactosidase, pustulanase and laminarinase had been detected. The level of DNA homology of three of the strains was 70-100%. The fourth isolate was genetically less related to the others (67% DNA relatedness) and phenotypically was more distant from other members of this group; however, all four strains were assigned to a single species genotypically. DNA from the strains isolated from mussels showed 40-45% genetic relatedness with the DNA of Alteromonas atlantica, 8-36% with DNA of Pseudoalteromonas haloplanktis subsp. haloplanktis, Pseudoalteromonas haloplanktis subsp. tetraodonis, Pseudoalteromonas undina, Pseudoalteromonas nigrifaciens and Pseudoalteromonasas carrageenovora, 53% with Pseudoalteromonas elyakovii, 32-48% with marine P. nigrifaciens from mussels and 14-16% with Alteromonas macleodii. The DNA-DNA hybridization data revealed that the levels of relatedness between the strains isolated and the type strains of Pseudoalteromonas citrea and Pseudoalteromonas fuliginea described recently were significant (95-85%). These results were confirmed by serological data employing polyclonal antibodies to cell surface antigens. The strains isolated from mussels were identified as P. citrea. The hybridization data showed that the name P. fuliginea Romanenko et al. 1994 should be recognized as a junior subjective synonym of P. citrea Gauthier 1977. A notable phenotypic diversity of P. citrea which might be a reflection of their ecological habitats is discussed.

MOLECULAR MODELLING OF ME2+ : (8-HYDROXY-QUINOLINATE)2 COMPLEXES USING ZINDO AND ESSF METHODS

The Me2+ (8-hydroxy-quinolinate)1-2 system was studied using semi-empirical (ZINDO) and molecular mechanics (ESFF) methods for a range of bivalent metals comprising alkaline earth metals (up to Sr2+) and the first two rows of transition metals. The structural validation of the optimisation calculations showed that ESFF is in general an efficient predictor of the structure of the complexes. On the other hand, ZINDO offers an appropriate tool for describing the mechanisms of complex formation for the studied system. The spectral validation based on the comparison of spectral data with predicted molecular geometric parameters shows that ZINDO offers a better tool for describing the mechanisms of complex formation. Finally, the consistent relationship (in relative terms) between SCF energies and equilibrium constants, and between computed charges at the oxygen ligation atom and pH, showed that ZINDO can be used as a tool for the design of chelating agents. For the system studied the CPU times are not prohibitive, even for a metal-comprehensive investigation.

Syntheses of trans-3′-substituted-CCG-IV analogs and their characterization to ionotropic glutamate receptors

Abstract Trans-3′-substituted-CCG-IV analogs ( 2 and 3 ) were efficiently synthesized via an intramolecular cycloaddition of diazoacetamide 4a using a chiral rhodium catalyst. These analogs evoked marked depolarization through ionotropic glutamate receptors on the spinal motoneurons or the kainate-sensitive dorsal root C-fiber of new born rats even though their binding affinities for the receptors on rat brain synaptic membranes were relatively low. These results suggest that the depolarizing action on C-fiber is not caused by the activation of kainate high affinity sites.

Design of stable α-helices using global sequence optimization

The rational design of peptide and protein helices is not only of practical importance for protein engineering but also is a useful approach in attempts to improve our understanding of protein folding. Recent modifications of theoretical models of helix‐coil transitions allow accurate predictions of the helix stability of monomeric peptides in water and provide new possibilities for protein design. We report here a new method for the design of α‐helices in peptides and proteins using AGADIR, the statistical mechanical theory for helix‐coil transitions in monomeric peptides and the tunneling algorithm of global optimization of multidimensional functions for optimization of amino acid sequences. CD measurements of helical content of peptides with optimized sequences indicate that the helical potential of protein amino acids is high enough to allow formation of stable α‐helices in peptides as short as of 10 residues in length. The results show the maximum achievable helix content (HC) of short peptides with fully optimized sequences at 5 °C is expected to be ∼70–75%. Under certain conditions the method can be a powerful practical tool for protein engineering. Unlike traditional approaches that are often used to increase protein stability by adding a few favorable interactions to the protein structure, this method deals with all possible sequences of protein helices and selects the best one from them. Copyright

Peroxidase-dependent fluorescence decrease of carboxyfluorescein and its enhancement by liposome encapsulation

The fluorescence intensity of 5(6)-carboxyfluorescein (CF) was decreased by addition of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The reaction inside a liposome containing CF and HRP on addition of H2O2 was measured fluorometrically after destruction of the liposome with Triton X-100. The reaction efficiency was higher than that without liposome because CF and HRP were concentrated inside the latter. The determination of H2O2 can be performed with a smaller amount of HRP by liposome encapsulation.

THE INFLUENCE OF THE HELICITY OF SOLUBLE PEPTIDES ON THEIR ADSORPTION KINETICS

Abstract The interfacial properties of aqueous solutions of two C-terminal (12–36) fragments of neuropeptide Y, with alanin or threonin as N-terminal amino-acid, have been studied by surface tension and surface potential measurements. The difference in the helicity between these two samples may explain the observed differences in their rates of surface tension decrease during the first 30xa0min of adsorption. Structural rearrangements at the solution/air interfaces following this initial step of adsorption resulted in the levelling of surface tension and surface potential data to the comparable values for both C-terminal fragments studied.

Molecular recognition using a peptide combinatorial library and its detection by surface plasmon resonance spectroscopy

Although peptides are potent molecules for specific molecular recognition of some small compounds, there are many difficulties in detecting these molecular complexes directly, since the affinity interactions are too small to detect without using specific labels, such as fluorescent probes. For this purpose, we developed a new methodology using surface plasmon resonance spectroscopy for sensitive detection of small molecules without using any label.