Purification and Some Properties of Nucleases from Shii-take,Lentinus edodes

Three kinds of nuclease preparations, each of which having both endonuclease activity that formed 5′-mononucleotides and 3′-nucleotidase activity, were separated and partially purified from Shii-take, Lentinus edodes. Both enzyme activities of each preparation showed a similar thermostability and electrophoretic mobility on Polyacrylamide gel, and a competitive relationship was observed between RNA and 3′-AMP in their enzyme reactions. From these results, it is concluded that both enzyme activities of these three preparations reside in a single protein, respectively. They resemble one another in substrate specificity, cleavage pattern of RNA and thermostability, but are distinguishable from one another by molecular weight, electrophoretic mobility and optimum pH for degradation of RNA.     

The structure of duplex DNA in the nucleosome core particle: An alternative model

Many studies indirectly indicate that the conformation ofin vivo duplex DNA is the double helix. The most direct view, from the X-ray analysis of the nucleosome core particle, has also been interpreted in terms of the double helix structure. However, an alternative possibility exists; that the duplex adopts a metastable side-by-side conformation which readily converts to the double helix on removal of protein. Evidence for the existence of this conformation has been obtained from a reanalysis of the electron density map for the nucleosome particle.     

New insights into the membrane topology of the phagocyte NADPH oxidase: characterization of an anti-gp91-phox conformational monoclonal antibody

Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.     

High resolution methylation analysis of the HoxA5 regulatory region in different somatic tissues of laboratory mouse during development

Homeobox genes encode a group of DNA binding regulatory proteins whose key function occurs in the spatial-temporal organization of genome during embryonic development and differentiation. The role of these Hox genes during ontogenesis makes it an important model for research. HoxA5 is a member of Hox gene family playing a central role during axial body patterning and morphogenesis. DNA modification studies have shown that the function of Hox genes is partly governed by the methylation-mediated gene expression regulation. Therefore the study aimed to investigate the role of epigenetic events in regulation of tissue-specific expression pattern of HoxA5 gene during mammalian development. The methodology adopted were sodium bisulfite genomic DNA sequencing, quantitative real-time PCR and chromatin-immunoprecipitation (ChIP). Methylation profiling of HoxA5 gene promoter shows higher methylation in adult as compared to fetus in various somatic tissues of mouse being highest in adult spleen. However q-PCR results show higher expression during fetal stages being highest in fetal intestine followed by brain, liver and spleen. These results clearly indicate a strict correlation between DNA methylation and tissue-specific gene expression. The findings of chromatin-immunoprecipitation (ChIP) have also reinforced that epigenetic event like DNA methylation plays important role in the regulation of tissue specific expression of HoxA5.     

Molecular mass and chain conformations of Rhizoma Panacis Japonici polysaccharides

The molecular mass and size of five water-soluble polysaccharides isolated from Rhizoma Panacis Japonici (RPJ) were determined with laser light scattering (LLS), size-exclusion chromatography (SEC) combined with LLS (SEC–LLS), dynamic light scattering (DLS), as well as transmission electron microscope (TEM). Their weight-average molecular masses (M_w) were 3.5 × 10⁴, 1.47 × 10⁵, 1.24 × 10⁶, 9.26 × 10⁵ and 1.36 × 10⁶, radii of gyration (<s²>_z^1/2) were 14.7, 31.7, 50.8, 41.8 and 40.4 nm, and hydrodynamic radii (R_h) were 19.9, 37.5, 66.2, 52.1, and 55.2 nm, respectively. The results showed that molecular masses and sizes of the polysaccharides were influenced by the pH and temperature of the extraction mediums. The conformation parameters were calculated from the above data according to polymer solution theory. The values of ρ (= <s²>_z^1/2/R_h) were from 0.7 to 0.8, exponents (ν) of <s²>_z^1/2 = k M_w^ν were from 0.31 to 0.43, and fractal dimension (d_f) were from 2.3 to 3.2, respectively. The results revealed that all of the polysaccharides existed as spheres in 0.15 M NaCl aqueous solution. TEM and atomic force microscope (AFM) further confirmed the spherical morphologies of these molecules. The spherical conformations of the polysaccharides were a result of their highly branched structures.     

A comparative NMR and molecular dynamics study of the conformations of bradykinin B1 and B2, B2, and B1-specific receptor antagonists B-9430, B-9436, and B-9858

Extensive proton magnetic resonance experiments were carried out on three bradykinin peptide antagonists B-9430, B-9436, and B-9858 in aqueous solutions as well as in sodium dodecylsulphate micelles (B-9430 and B-9436) and CD₃OH/H₂O (60%/40%) mixtures for B-9858. All three peptides showed no observable secondary structure in aqueous solution. However, in their respective structure-inducing solvents, B-9430 (B₁ and B₂ receptor antagonist) and B-9436 (a B₂ receptor antagonist) exhibit a type II β-turn involving residues 2–5, and B-9430 also exhibits a type II′ β-turn involving residues 6–9 (in sodium dodecylsulfate micellar solutions), whereas B-9858, a B₁-specific receptor antagonist, exhibits only a type II β-turn involving residues 2–5 (in CD₃OH/H₂O solutions). Simulated annealing calculations on B-9858 confirm the experimental conclusions based on the nmr data. In addition, simulated annealing of the (2S, 3aS, 7aS)-octahydroindole-2-carboxylic acid (Oic residue), which is present in two of the three decapeptides studied, show that the one-chair conformation of the six-membered ring predominates, in agreement with the experimental data. The activities of these peptides are compared with their secondary structures and the specific receptor activity appears to depend on the presence of specific amino acid residues, such as N-(2-indanyl)glycine (Nig) and D[α-(2-indanyl)glycine] (D-Igl) as well as on elements of secondary structure. © 1997 John Wiley & Sons, Inc. Biopoly 42: 521–535, 1997     

NMR studies of dextromethorphan in both isotropic and anisotropic states

Herein, we present the solution‐state NMR studies on dextromethorphan ( 1 ) under both isotropic and anisotropic conditions. From the measurement of 22 residual dipolar couplings using a stretched polystyrene gel (PS), we show that accurate and detailed structural information is readily determined including the relative stereochemical assignments of chiral centers, validation of diastereomer configuration, and the stereospecific assignment of the seven pairs of prochiral protons. This utility of PS gels is thus showcased to obtain rapid, accurate conformational, and relative configuration information in this important class of compounds without recourse to X‐ray analysis. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

β‐Hairpin stabilization in a 28‐residue peptide derived from the β‐subunit sequence of human chorionic gonadotropin hormone

The β‐subunit of the human chorionic gonadotropin (hCG) hormone, which is believed to be related to certain types of cancer, contains three hairpin‐like fragments. To investigate the role of β‐hairpin formation in the early stages of the hCGβ folding, a 28‐residue peptide with the sequence RDVRFESIRLPGSPRGVNPVVSYAVALS, corresponding to the H3‐β hairpin fragment (residues 60–87) of the hCGβ subunit, was studied under various conditions using three optical spectroscopic methods: Fourier transform ir spectroscopy, electronic CD, and vibrational CD. Environmental conditions are critical factors for formation of secondary structure in this peptide. TFE : H₂O mixed solvents induced helical formation. Formation of β‐structure in this peptide, which may be related to the native β‐hairpin formation in the intact hormone, was found to be induced only under conditions such as high concentration, high temperature, and the presence of nonmicellar sodium dodecyl sulfate concentrations. These findings support a protein folding mechanism for the hCGβ subunit in which an initial hydrophobic collapse, which increases intermolecular interactions in hCGβ, is needed to induce the H3‐β hairpin formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 413–423, 1999     

Effect of viscosity and dielectric constant variation on fractional fluorescence quenching analysis of coumarin dye in binary solvent mixtures

Photo physical properties of fluorescent organic compounds give an immense improved knowledge on characteristics of excited state that is beneficial to devise innovate molecules and understand their performance in particular applications. Coumarin derivatives have been extensively investigated in this regard. This article narrates steady state fluorescence quenching measurements of a coumarin derivative namely 3‐hydroxy‐3‐[2‐oxo‐2‐(3‐oxo‐3H‐benzo[f]chromen‐2‐yl)‐ethyl]‐1,3‐dihydro‐indol‐2‐one (3HBCD) in a binary mixture of acetonitrile and 1,4‐dioxane. Aniline is used as quencher. Fluorescence intensity is large in acetonitrile and decreases as the percentage of 1,4‐dioxane in the solvent mixture increases. With modest quencher concentration a deviation towards the x axis is noticed in the Stern–Volmer (S–V) plots. This downward curvature is interpreted as due to the presence of 3HBCD in different conformers in the lowest energy level. Ground state intramolecular hydrogen bonding formation is observed due to the conformational changes in the solute. Figured estimations of various quenching parameters recommend that, while dynamic quenching prompts linearity in S–V plot at lower quencher concentration, increasing quenching efficiency with increasing medium viscosity suggests that reaction is not entirely controlled by material diffusion. Stern–Volmer constant increases with decreasing medium dielectric constant.     

Conformation and ion channel properties of a five-helix Bundle protein.

The primary amphipathic peptide Ac-Met-Gly-Leu-Gly-Leu-Trp-Leu-Leu-Val-Leu10-Ala-Ala-Ala-Leu-Gln-Gly-Ala-Lys-Lys-Lys20-Arg-Lys-Val-NH-CH2-CH2-SH called SPM was able to induce formation of ion channels into planar lipid bilayers with main conductance values of 75 and 950 pS in 1 M KCl. The 75 pS value can be attributed to an aggregate composed of five monomers since the corresponding five-unit bundle (5-SPM) also presented a 70 pS channels under the same conditions. The upper 950 pS level would be generated by a hexameric aggregate. Ion channels induced by both SPM and its pentameric bundle are slightly cation selective but not voltage-dependent. The structural studies showed that the SPM and 5-SPM possess mainly an alpha-helical structure (approximately 40%) and are strongly embedded in the bilayer. This behaviour and the strong hydrophobic interactions occurring between helices in the bundle induce a strong stabilization of 5-SPM in the bilayer and would be responsible for the stepwise current fluctuations observed during the incorporation of 5-SPM into the membrane.