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tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5ʹ-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5ʹ-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 A resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5ʹ-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.

Many animals show left-right (LR) asymmetric morphology. The mechanisms of LR asymmetric development are evolutionarily divergent, and they remain elusive in invertebrates. Various organs in Drosophila melanogaster show stereotypic LR asymmetry, including the embryonic gut. The Drosophila embryonic hindgut twists 90° left-handedly, thereby generating directional LR asymmetry. We recently revealed that the hindgut epithelial cell is chiral in shape and other properties; this is termed planar cell chirality (PCC). We previously showed by computer modeling that PCC is sufficient to induce the hindgut rotation. In addition, both the PCC and the direction of hindgut twisting are reversed in Myosin31DF (Myo31DF) mutants. Myo31DF encodes Drosophila MyosinID, an actin-based motor protein, whose molecular functions in LR asymmetric development are largely unknown. Here, to understand how PCC directs the asymmetric cell-shape, we analyzed PCC in genetic mosaics composed of cells homozygous for mutant Myo31DF, some of which also overexpressed wild-type Myo31DF. Wild-type cell-shape chirality only formed in the Myo31DF-overexpressing cells, suggesting that cell-shape chirality was established in each cell and reflects intrinsic PCC. A computer model recapitulating the development of this genetic mosaic suggested that mechanical interactions between cells are required for the cell-shape behavior seen in vivo. Our mosaic analysis also suggested that during hindgut rotation in vivo, wild-type Myo31DF suppresses the elongation of cell boundaries, supporting the idea that cell-shape chirality is an intrinsic property determined in each cell. However, the amount and distribution of F-actin and Myosin II, which are known to help generate the contraction force on cell boundaries, did not show differences between Myo31DF mutant cells and wild-type cells, suggesting that the static amount and distribution of these proteins are not involved in the suppression of cell-boundary elongation. Taken together, our results suggest that cell-shape chirality is intrinsically formed in each cell, and that mechanical force from intercellular interactions contributes to its formation and/or maintenance.

Elucidating the global and local rules that govern genome-wide, hierarchical chromatin architecture remains a critical challenge. Current high-throughput chromosome conformation capture (Hi-C) technologies have identified large-scale chromatin structural motifs, such as topologically associating domains and looping. However, structural rules at the smallest or nucleosome scale remain poorly understood. Here, we coupled nucleosome-resolved Hi-C technology with simulated annealing-molecular dynamics (SA-MD) simulation to reveal 3D spatial distributions of nucleosomes and their genome-wide orientation in chromatin. Our method, called Hi-CO, revealed distinct nucleosome folding motifs across the yeast genome. Our results uncovered two types of basic secondary structural motifs in nucleosome folding: α-tetrahedron and β-rhombus analogous to α helix and β sheet motifs in protein folding. Using mutants and cell-cycle-synchronized cells, we further uncovered motifs with specific nucleosome positioning and orientation coupled to epigenetic features at individual loci. By illuminating molecular-level structure-function relationships in eukaryotic chromatin, our findings establish organizational principles of nucleosome folding.

Hydrodynamic interactions (HI) give rise to collective motions between molecules, which are known to be important in the dynamics of random coil polymers and colloids. However, their role in the biological self-assembly of many molecule systems has not been investigated. Here, using Brownian dynamics simulations, we evaluate the importance of HI on the kinetics of self-assembly of lipid membranes. One-thousand coarse-grained lipid molecules in periodic simulation boxes were allowed to assemble into stable bilayers in the presence and absence of intermolecular HI. Hydrodynamic interactions reduce the monomer-monomer association rate by 50%. In contrast, the rate of association of lipid clusters is much faster in the presence of intermolecular HI. In fact, with intermolecular HI, the membrane self-assembly rate is 3–10 times faster than that without intermolecular HI. We introduce an analytical model to describe the size dependence of the diffusive encounter rate of particle clusters, which can qualitatively explain our simulation results for the early stage of the membrane self-assembly process. These results clearly suggest that HI greatly affects the kinetics of self-assembly and that simulations without HI will significantly underestimate the kinetic parameters of such processes.

The effect of macromolecular crowding on protein diffusion has been extensively studied in order to understand the protein dynamics in cellular environments. Previous studies have generally focused on proteins as crowding agents as they are known to occupy 20-30% of the cell volume. The volume fraction of the nucleoid in bacterial cells is also significant, around 10-20%, but the effect of chromosomal DNA as a crowding agent on protein diffusion has drawn relatively limited attention. Using recently build high-resolution models of bacterial nucleoids, Brownian and Stokesian dynamics simulations of coarse-grained model systems containing chromosomal DNA and proteins were carried out to investigate the effect of nucleoid crowding on protein diffusion. The coarse-grained model for the nucleoid is an experimentally-derived 3D model of the Caulobacter crescentus chromosome at base-pair resolution built by combining topological information of chromosomal DNA with distance restraints obtained from high-throughput Chromosome Conformation Capture (HiC) experiments, while proteins in the systems are modeled as hard-spheres. Results show that diffusion of proteins are slower around the nucleoid compared to in protein crowding and that there is size-dependent exclusion of proteins from the chromosome interior. Further analysis was carried to examine the degree of anomalous diffusion at different time and length scales.

Interlaminar fracture toughness and delamination fatigue crack growth behavior were investigated for carbon fiber (CF)/epoxy laminates with the self-same epoxy interleaf. The matrix epoxy with a thickness of 50 μm was chosen as the interleaf material in order to clarify the effect of resin-rich layer thickness on the delamination fatigue crack growth behavior. Tests under mode I loading were carried out using double cantilever beam specimens. For tests under mode II loading, three-point end notched flexure specimens were used for interlaminar fracture toughness tests, while four-point end notched flexure specimens were used for delamination fatigue tests. The mode I properties (interlaminar fracture toughness and delamination fatigue threshold) of these epoxy-interleaved CFRP laminates were almost identical to those of the laminates without interleaf (base CFRP laminates). The effect of epoxy interleaf was completely different under mode II loading. The mode II interlaminar fracture toughness for the epoxy-interleaved laminates was 1.6 (initial value) and 3.4 (propagation value) times higher than that for the base CFRP laminates. The mode II delamination fatigue threshold of the epoxy-interleaved laminates was 2–2.3 times higher than those of the base CFRP laminates. While the toughness of the interleaf is the key factor under mode I, the thickness of interlayer is the key factor under mode II. The difference in the effect of the self-same epoxy interlayer on the interlaminar fracture properties under modes I and II loadings was discussed on the bases of the fractographic observations and mechanism considerations.

BACKGROUND Although transurethral resection of a bladder tumor (TUR-Bt) alone has been standard treatment for single superficial bladder carcinoma, some authors reported a certain prophylactic effect of a single immediate intravesical instillation of chemotherapeutic agent after TUR-Bt. A prospective randomized study was conducted to determine whether a single (2″R)-4′-O-tetrahydropyranyl-doxorubicin (THP) instillation immediately after TUR-Bt is beneficial to patients with a single superficial bladder carcinoma. METHODS One hundred seventy patients with a single resectable superficial bladder carcinoma (Ta-1, primary or recurrent with no recurrence during the last 1 year) were enrolled in this study. THP (30 mg/30 mL of normal saline) was administered into the bladder within 6 hours after TUR-Bt in arm A, while TUR-Bt alone was done in arm B. RESULTS Of the 170 patients, 160 (94.1%) were eligible and were followed up for a median time of 40.8 months. There was a significant difference in the recurrence free curve between the 2 arms (log-rank test; P = 0.0026), with 92.4% recurrence free rate at 1 year, 82.7% at 2 years, and 78.8% at 3 years in arm A (84 patients) and 67.0%, 55.7%, and 52.6%, respectively, in arm B. The recurrence rate per year was 0.11 ± 0.22 in arm A and 0.24 ± 0.36 in arm B, with a significant difference (P = 0.007). Toxicity included pain with micturition in 9 patients (10.7%), urinary frequency/urgency in 5 patients (6.0%), and macroscopic hematuria in 7 patients (8.3%). CONCLUSIONS These data indicate that a single THP instillation immediately after TUR reduces the recurrence of superficial bladder carcinoma. Cancer 2002;94:2363–8. © 2002 American Cancer Society. DOI 10.1002/cncr.10496

Self-organization is one of the most fundamental phenomena in biology. Elucidating the mechanisms of the self-assembly of biological molecules, such as protein folding/binding and formation of lipid bilayer membranes, have been a fascinating topic over decades in biological field. Hydrodynamic interactions (HIs) give rise to collective motions between molecules. Therefore, they may play an important role on dynamics of self-assembly. Although there are significant advances in theoretical and computational studies on thermodynamics and kinetics of self-assembling systems recent years, analysis on effects of HIs to the reactions are limited due to their high complexity. Here, by using a Brownian dynamics (BD) simulation technique, we evaluate the importance of HIs on kinetic of self-assembly in a set of examples, formation of lipid membrane and actin polymerization reaction. We first built coarse-grained models of lipid and actin monomer. A cluster of lipid molecules was represented by two particles, one is hydrophilic head group and the other is hydrophobic tail group, and attractive interaction between tail groups was applied. For actin polymerization system, each monomer was represented by two particles and an angle-dependent attractive interaction was applied to form long filaments. In BD simulations, the Rotne-Prager-Yamakawa tensor was used to account for HIs between particles. In the early stage of self-assembly, HIs between inter-monomer particles decelerated the association rate of monomers to form oligomers compared with that in the simulations without inter-monomer HIs in both systems. On the other hand, the HIs accelerate the reaction of oligomers to form a fully-assembled state or much longer polymers. This phenomenon could be explained by the Kirkwood-Riseman theory. Those results clearly suggest that HIs greatly affect kinetics of self-assembly reactions and considering the interactions is crucial for studying dynamics of biological systems.