Atomic-level characterization of free volume in the structure of Cu67Zr33amorphous alloy.

The structure of Cu67Zr33amorphous alloy was investigated in terms of packing density and free volume by using neutron, x-ray diffraction and reverse Monte Carlo (RMC) modelling. The RMC model was analysed by a method of decomposing the three-dimensional atomic configuration into fundamental polyhedral units (termed as 'holes' referencing the Bernal's works) of which faces are all triangles consisting of chemical bonds. Not only tetrahedral and octahedral holes but also other larger holes were identified. Moreover, the atomic packing fractions and free volumes in the respective polyhedral holes were evaluated with reference to those for the corresponding crystal structures. The results show that the distribution of free volumes for the larger holes can be described by the exponential function assuming that there are no energetic interactions between each other. On the other hand, the local structural fluctuations due to densely and loosely packed tetrahedral holes were observed, leading to the negative free volume spaces.

Observation of High-Frequency Transverse Phonons in Metallic Glasses.

Using inelastic neutron scattering and molecular dynamics simulations on a model Zr-Cu-Al metallic glass, we show that transverse phonons persist well into the high-frequency regime, and can be detected at large momentum transfer. Furthermore, the apparent peak width of the transverse phonons was found to follow the static structure factor. The one-to-one correspondence, which was demonstrated for both Zr-Cu-Al metallic glass and a three-dimensional Lennard-Jones model glass, suggests a universal correlation between the phonon dynamics and the underlying disordered structure. This remarkable correlation, not found for longitudinal phonons, underscores the key role that transverse phonons hold for understanding the structure-dynamics relationship in disordered materials.

Formation of NaCl-type monodeuteride LaD by the disproportionation reaction of LaD2.

Previous x-ray diffraction measurements revealed the pressure-induced decomposition of an fcc LaH2.3 into H-rich and H-poor fcc phases around 11 GPa. The present neutron diffraction measurements on LaD2 confirm the formation of NaCl-type LaD as a counterpart of the D-rich LaD2+δ by disproportionation. First-principles enthalpy and lattice dynamic calculations demonstrate that the NaCl-type LaH is stabilized at high pressures and can be recovered at ambient conditions. Finding the NaCl-type LaH will pave the way for investigations on the site-dependent nature of hydrogen-metal interactions.

Examination of gas desorption by B4C resin for use in neutron scattering experiment.

Gas desorption rates for several types of B(4)C resins were investigated using a throughput method. The investigation was particularly focused on determining the out gas composition, effects of dry air, grain size (density) effects on the gas desorption rates. It is found that water is the main component of out gas and that dry air can effectively reduce gas desorption.

A prospective, double-blind, randomized trial of caudal block using ropivacaine 0.2% with or without fentanyl 1 microg kg-1 in children.

BACKGROUND: It has been reported that ropivacaine produces vasoconstriction in contrast to vasodilation produced by bupivacaine. It is possible that additives to ropivacaine can provide further analgesic advantages compared with bupivacaine. We thus evaluated whether the addition of fentanyl to ropivacaine prolonged the duration of analgesia after a single shot caudal block. METHODS: A total of 36 children undergoing surgical procedures below the umbilicus were randomly allocated to one of two groups: Group F received ropivacaine 0.2%, 1 ml kg(-1) with fentanyl 1 microg kg(-1) and Group S received ropivacaine 0.2%, 1 ml kg(-1) with saline. The analgesic effect of the caudal block was evaluated using the Children's Hospital of Eastern Ontario Pain Scale (CHEOPS) and sedation was assessed using the Steward score at 30 min after extubation and at 1, 2, 4, 6, 12 and 24 h. The first analgesic requirement time and side-effects in a 24 h period were also recorded. RESULTS: There were no differences in characteristics between the groups. The end-tidal concentration of sevoflurane at extubation in Group F was significantly lower than in Group S. However, there was no significant difference in time from discontinuation of the volatile anaesthetics to tracheal extubation. No statistical differences were found in the CHEOPS and Steward score, and the time to first analgesia. The incidence of postoperative vomiting was not significantly different. CONCLUSION: We found that the addition of fentanyl 1 mug kg(-1) to ropivacaine 0.2% for caudal analgesia provides no further analgesic advantages over ropivacaine 0.2% alone.

Mass-fractal clustering and power-law decay of cluster size in 1-propanol aqueous solution.

Mesoscale structure of 1-propanol aqueous solutions with propanol mole fraction xp ranging from 0.1 to 0.33 has been studied by means of small angle neutron scattering (SANS) and large-scale reverse Monte Carlo (RMC) technique. Analysis of the SANS intensities in terms of a fractal model shows that the fractal dimension df of mesoscale structure of the solution is about 1.8-1.9 for water-rich solution and about 1.5 for propanol-rich solution. Percolation analysis on the RMC results reveals that the water molecules and the propanol molecules cluster, respectively, as a mass fractal, the dimension dM of which is about 2.3-2.5 for both clusters for water-rich solution. Furthermore, the distribution of the cluster size is expressed by a simple power law with an exponent tau of about 1.35-1.5 for the propanol clusters and 1.05-1.2 for the water clusters. These results imply that the current solution is characterized by polydisperse mass fractals. In fact, a theoretical relation for polydisperse system of mass fractals, df = dM(2-tau), holds well in the current solution. The characteristic change in df from 1.8-1.9 to 1.5 described above is attributed to the crossover between the water-rich regime and the propanol-rich regime. Most of the water molecules and the propanol molecules are located on the interface between clusters, and the water molecules form thin layers of about 10 A thick irrespective of 1-propanol content studied.

EBV replicon vector system enhances transgene expression in vivo: applications to cancer gene therapy.

BACKGROUND: A major limitation of current cancer gene therapies is low and transient expression of the therapeutic gene. For long-term expression of transgenes in vivo, an Epstein-Barr virus (EBV) replicon vector has been developed. The present study examines the effect of the EBV replicon vector system and its application to a suicide gene therapy for melanoma in mice. METHODS: An EBV replicon vector system, pEBc, consisting of EBV nuclear antigen-1 (EBNA-1) and the origin of latent viral DNA replication, oriP, was used to express either the luciferase gene or the herpes simplex virus (HSV) thymidine kinase (TK) gene. The expression vector was introduced in vivo into melanoma tumor masses in mice by means of HVJ-cationic liposomes. The time-course of gene expression and the anticancer effect of the EBV replicon vector were investigated in comparison with pcLuc, which lacks the EBV components. RESULTS: Luciferase expression was sustained in both cultured cells and melanoma masses by pEBc but not by pcLuc. The luciferase expression level in melanoma masses was higher by pEBcLuc than by pcLuc, although Southern blot analysis showed the number of copies of pEBcLuc retained in the melanoma masses to be fewer than that of pcLuc. The effectiveness of EBV replicon vector on suicide gene therapy of melanoma in mice was also demonstrated. CONCLUSION: The EBV replicon vector appears useful for cancer gene therapy. Analysis of the transgene in tumors suggests that the EBV replicon system may be responsible for efficient transcription but not retention of the transgene.

Structure of the heterodimeric complex between CAD domains of CAD and ICAD.

We present here the structure of the complex between the CAD domain of caspase activated deoxyribonuclease (CAD) and the CAD domain of its inhibitor (ICAD), determined by nuclear magnetic resonance spectroscopy. The two domains adopt a very similar fold, which consists of an alpha-helix and a beta-sheet, and are aligned side by side in the complex. Notably, the positive charges on the strand beta2 at one end of the beta-sheet of CAD and negative charges around the opposite end of the beta-sheet of ICAD are paired in the complex. Point mutations of the charged amino acids at this interface, on either CAD or ICAD, prevented formation of the functional CAD-ICAD complex. This implies that the interaction between the CAD domains of CAD and ICAD is an essential step in the correct folding of CAD in the complex.

Structural study of the N-terminal domain of the alpha subunit of Escherichia coli RNA polymerase solubilized with non-denaturing detergents.

The amino-terminal domain of the alpha subunit (alphaNTD) of Escherichia coli RNA polymerase consisting of 235 amino acid residues functions in the assembly of the alpha, beta, and beta' subunits into the core-enzyme. It has a tendency to form aggregates by itself at higher concentrations. For NMR structural analysis of alphaNTD, the solution conditions, including the use of non-denaturing detergents, were optimized by monitoring the translational diffusion coefficients using the field gradient NMR technique. Under the optimal conditions with taurodeoxycholate and with the aid of deuteration of the sample, alphaNTD gave triple-resonance spectra of good quality, which allowed the assignment of a large part of the backbone resonances. Analysis of the pattern of NOEs observed between the backbone amide and alpha-protons demonstrated that alphaNTD has three alpha-helices and two beta-sheets. Although the secondary structure elements essentially coincide with those in the crystal structure, the larger of the two beta-sheets has two additional beta-strands. The irregular NOE patterns observed for the three positions in the beta-sheets suggest the presence of beta-bulge structures. The positions of the three helices coincide with the conserved sequence regions that are responsible for the subunit assembly.

Structure of the CAD domain of caspase-activated DNase and interaction with the CAD domain of its inhibitor.

Caspase-activated DNase (CAD), which causes a genome fragmentation at the final stage of apoptosis, is a protein of about 40 kDa and exists as a complex form with the inhibitor ICAD in living cells. There is sequence homology of about 80 amino acid residues at the N termini of CAD and ICAD (called the CAD domain). Here, we report the three-dimensional structure of the CAD domain of CAD determined by multi-dimensional NMR spectroscopy and the property of CAD domains investigated by a surface plasmon resonance experiment. The CAD domain of CAD is an independently folded domain composed of one alpha-helix and five beta-strands forming a single sheet. The overall structure is categorized in the ubiquitin superfold. This domain can bind strongly to the isolated CAD domain of ICAD (dissociation constant: 5.48(+/-0.003)x10(-8) M). It suggests the function of the CAD domains in the CAD-ICAD system, that the protein-protein interaction through the CAD domains plays an important role in the inhibition of CAD DNase activity and in the correct folding of CAD. On the basis of structural comparison with other protein complexes containing the ubiquitin superfold, the interaction mode of the CAD domains is proposed.

Synthesis and properties of a new type DNA dendrimer.

We have designed a new type of a DNA dendrimer which has rigid branched structure. The branching molecule was prepared from 1,3,5-tribromobenzene. The dendrimer unit, in which three oligonucleotide-chains, two molecules of T15 and one molecule of A15, linked to the branching molecule, was synthesized by an automated DNA synthesizer. The properties of the dendrimer unit and dendrimer formation by inter-molecular association of T15 and A15 chains of the dendrimer unit will be presented.

NMR observation of selected segments in a larger protein: central-segment isotope labeling through intein-mediated ligation.

Peptide segments in a protein, which can include an active site of interest or be a series of parts constituting the entire structure, are now selectively observed by nuclear magnetic resonance (NMR) spectroscopy using samples prepared by the intein-mediated ligation method. Two separate inteins were used to ligate NMR-transparent segments to both the ends of an NMR-visible segment, producing a partly visible intact protein molecule. The (15)N-(1)H correlation spectrum of a 370-residue maltose binding protein labeled with (15)N at a continuous segment comprising residues Gly(101)-Ser(238) showed the essential elimination of signal overlapping, the signals being at the same positions as for the uniformly labeled sample. This method will allow structural analysis by NMR of over 50-kDa proteins in combination with contemporary NMR techniques suppressing the signal decays of larger proteins.

Improved segmental isotope labeling of proteins and application to a larger protein.

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591-5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13C/15N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.

Flexible linker in the RNA polymerase alpha subunit facilitates the independent motion of the C-terminal activator contact domain.

The dynamic properties of the C-terminal one-third of the alpha subunit of RNA polymerase were investigated. The intact alpha subunit exhibited almost the same NMR spectral pattern as the isolated C-terminal fragment, indicating that the C-terminal domain retains the same conformation as the isolated fragment, and that its motion is independent of that of the associated N-terminal domain. Analysis of the NMR dynamics data for the intact alpha subunit indicated that at least 13 residues between the N and C-terminal domains show distinctly higher motional flexibility than the structured parts. This flexible linker may endow the C-terminal domain with locational freedom in different kinds of initiation complex. The dynamics data also revealed that the residues in the contact site for DNA and transcription factors exhibited higher mobility than other secondary structural elements.