Single-molecule imaging of the dynamic interactions between macromolecules

In recent years, the development of single-molecule detection techniques has allowed the dynamic properties of biomolecules, which are normally obscured in conventional ensemble measurements, to be measured. One of these single-molecule detection techniques allows the measurement of dissociation and association events of individual molecules to be measured. This technique is based on the unique premise that the mobility between molecules that are bound and the mobility between those that are free in solution are different. The binding of ATP at the beginning and its dissociation at the end of the hydrolysis reaction were detected at the single-molecule level in real time. In this study, we extended this technique to image the dynamic interactions between large biomolecules (protein/protein and protein/polysaccharide). The binding and dissociation of fluorescently labeled macromolecules to partner molecules fixed on a glass surface were visualized by total internal reflection fluorescence microscopy. The dynamic interactions between the proteins in two energy conversion systems, that is, signaling proteins and enzyme molecules moving on dextran, have been measured. In these systems, the dynamic interactions were sensitive to the factors determining the chemical reactions. Thus, the dynamic interactions monitored in the single-molecule measurements provided useful information to further the understanding of the underlying mechanisms of energy conversion systems.     

High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling

Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.     

Cognitive map formation through sequence encoding by theta phase precession

The rodent hippocampus has been thought to represent the spatial environment as a cognitive map. The associative connections in the hippocampus imply that a neural entity represents the map as a geometrical network of hippocampal cells in terms of a chart. According to recent experimental observations, the cells fire successively relative to the theta oscillation of the local field potential, called theta phase precession, when the animal is running. This observation suggests the learning of temporal sequences with asymmetric connections in the hippocampus, but it also gives rather inconsistent implications on the formation of the chart that should consist of symmetric connections for space coding. In this study, we hypothesize that the chart is generated with theta phase coding through the integration of asymmetric connections. Our computer experiments use a hippocampal network model to demonstrate that a geometrical network is formed through running experiences in a few minutes. Asymmetric connections are found to remain and distribute heterogeneously in the network. The obtained network exhibits the spatial localization of activities at each instance as the chart does and their propagation that represents behavioral motions with multidirectional properties. We conclude that theta phase precession and the Hebbian rule with a time delay can provide the neural principles for learning the cognitive map.     

Analysis on split failure of cladding of high burnup BWR rods in reactivity-initiated accident conditions by RANNS code

A computer code RANNS was developed to analyze fuel rod behaviors in the reactivity-initiated accident (RIA) conditions. RANNS performs thermal and finite-element mechanical calculation for a single rod in axis-symmetric geometry, where fuel pellet consists of 36 equal-volume ring elements and cladding metallic wall consists of eight equal-thickness ring elements and one outer oxide element. The code can calculate temperature profile inside the rod, contact pressure generated by pellet–clad mechanical interaction (PCMI), stress–strain distribution and their interactions elaborately. An experimental analysis by RANNS begins with pre-test conditions of irradiated rod which are given by the fuel performance code FEMAXI-6.In the present study, analysis was performed on the simulated RIA experiments in the “nuclear safety research reactor” (NSRR), FK-10 and FK-12, with high burnup BWR rods in a cold-start up condition, and stress–strain evolution in the PCMI process was calculated extensively. In the analysis, the pellet–clad bonding was assumed both in the heat conduction and in mechanical restraint. The calculated hoop strain increase was compared with the measured strain gauge data, and satisfactory agreement was obtained. Simulation calculations with broader power pulses anticipated in RIA of commercial BWR were carried out and the resulted cladding hoop stress was compared with the failure stress estimated by comparison of analysis with experimental data.     

High quality gate insulator film formation on SiC using by microwave-excited high-density plasma

High quality gate insulator film formation on 4H-SiC substrate is demonstrated. The insulator films were formed by the PECVD and radical oxynitridation using microwave-excited high-density plasma with NO gas at low temperature. The oxide fixed charge and the interface trap density can be dramatically reduced by NO gas radical oxynitridation after the oxide film formed by the PECVD compared with by direct oxynitridation on 4H-SiC. SIMS profiles show carbon profile in these fabricated gate insulator films. It is confirmed that the electrical property is improved as the amount of remaining carbon in the insulator film decreases.     

Production of optically active 1,2,4-butanetriol from corresponding racemate by Microbial stereoinversion

Sterigmatomyces elviae DSM 70852 produced 12 g/l (S)-1,2,4-butanetriol (enantiomeric excess >99.9%) from 20 g/l racemate in 82 h. From the results of the inversion of an (R)-isomer to an (S)-isomer and GC-MS, it was suggested that (R)-1,2,4-butanetriol is oxidized to 1,4-dihydroxy-2-butanone, which is reduced to an (S)-isomer.     

Design of gem‐Difluoro‐bis‐Tetrahydrofuran as P2 Ligand for HIV‐1 Protease Inhibitors to Improve Brain Penetration: Synthesis,X‐ray Studies,and Biological Evaluation

The structure‐based design, synthesis, biological evaluation, and X‐ray structural studies of fluorine‐containing HIV‐1 protease inhibitors are described. The synthesis of both enantiomers of the gem‐difluoro‐bis‐THF ligands was carried out in a stereoselective manner using a Reformatskii–Claisen reaction as the key step. Optically active ligands were converted into protease inhibitors. Two of these inhibitors, (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐methoxyphenyl)sulfonamido)‐1‐phenylbutan‐2‐yl) carbamate ( 3 ) and (3R,3aS,6aS)‐4,4‐difluorohexahydrofuro[2,3‐b]furan‐3‐yl(2S,3R)‐3‐hydroxy‐4‐((N‐isobutyl‐4‐aminophenyl)sulfonamido)phenylbutan‐2‐yl) carbamate ( 4 ), exhibited HIV‐1 protease inhibitory K_i values in the picomolar range. Both 3 and 4 showed very potent antiviral activity, with respective EC₅₀ values of 0.8 and 3.1 nM against the laboratory strain HIV‐1_LAI. The two inhibitors exhibited better lipophilicity profiles than darunavir, and also showed much improved blood–brain barrier permeability in an in vitro model. A high‐resolution X‐ray structure of inhibitor 4 in complex with HIV‐1 protease was determined, revealing that the fluorinated ligand makes extensive interactions with the S2 subsite of HIV‐1 protease, including hydrogen bonding interactions with the protease backbone atoms. Moreover, both fluorine atoms on the bis‐THF ligand formed strong interactions with the flap Gly 48 carbonyl oxygen atom.     

Droplet‐Shooting and Size‐Filtration (DSSF) Method for Synthesis of Cell‐Sized Liposomes with Controlled Lipid Compositions

We report a centrifugal microfluidic method, droplet‐shooting and size‐filtration (DSSF), for the production of cell‐sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil‐water interface, thus resulting in the generation of cell‐sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5–2 μL) of sample solution is required for the formation of hundreds of cell‐sized liposomes. We believe that this method can be applied to generate cell‐sized liposomes for a wide variety of uses, such as the construction of artificial cell‐like systems.     

Design,Synthesis and X-Ray Structural Studies of Potent HIV-1 Protease Inhibitors Containing C-4 Substituted Tricyclic Hexahydro-Furofuran Derivatives as P2 Ligands

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2′ ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2′ ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high-resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.     

Synthesis and characterization of poly(vinyl alcohol) carbonate

Poly(vinyl alcohol) (PVA) was reacted with dimethyl carbonate (DMC) to prepare PVA carbonate (PVAC), which is considered to have three structures, cyclic carbonate (I), monocarbonate (II) and crosslinking carbonate (III). Though the reaction proceeded easily in dimethyl sulfoxide at 120 °C without any catalyst, the yield of the ethanol‐insoluble part of the products was less than that with the reaction containing a catalyst such as tetrabutylammonium bromide (TBAB). TBAB concentration did not affect the rate of carbonic esterification but affected the yield of the ethanol‐insoluble part. The rate of carbonic esterification increased with an increase in reaction temperature, and the activation energy of this reaction was 114 kJ mol⁻¹. Carbonic esterification under reduced pressure showed a larger degree of esterification than that of the reaction under atmospheric pressure. PVAC was soluble in water at a low degree of carbonic esterification (DC) but became insoluble as DC increased. The thermal property of PVA reduced by the carbonic esterification was improved as DC increased. © 2000 Society of Chemical Industry