Coarse-grained Modeling and Simulation of Actin Filament Behavior Based on Brownian Dynamics Method

The actin filament, which is the most abundant component of the cytoskeleton, plays important roles in fundamental cellular activities such as shape determination, cell motility, and mechanosensing. In each activity, the actin filament dynamically changes its structure by polymerization, depolymerization, and severing. These phenomena occur on the scales ranging from the dynamics of actin molecules to filament structural changes with its deformation due to the various forces, for example, by the membrane and solvent. To better understand the actin filament dynamics, it is important to focus on these scales and develop its mathematical model. Thus, the objectives of this study were to model and simulate actin filament polymerization, depolymerization, and severing based on the Brownian dynamics method. In the model, the actin monomers and the solvent were considered as globular particles and a continuum, respectively. The motion of the actin molecules was assumed to follow the Langevin equation. The polymerization, which increases the filament length, was determined by the distance between the center of the actin particle at the barbed end and actin particles in the solvent. The depolymerization, which decreases the filament length, was modeled such that the number of dissociation particles from the filament end per unit time was constant. In addition, the filament severing, in which one filament divides into two, was modeled to occur at an equal rate along the filament. Then, we simulated the actin filament dynamics using the developed model, and analyzed the filament elongation rate, its turnover, and the effects of filament severing on the polymerization and depolymerization. Results indicated that the model reproduced the linear dependence of the filament elongation on time, filament turnover process by polymerization and depolymerization, and acceleration of the polymerization and depolymerization by severing, which qualitatively agreed with those observed in experiments.     

Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme

DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp. KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, when long distance PCR (>5 kbp) was performed with KOD DNA polymerase, amplification efficiency (product yield) becomes lower because of its strong 3'-5' exonuclease activity for proof-reading. In order to improve a target length limitation in PCR, mutant DNA polymerases with decreased 3'-5' exonuclease activity were designed by substituting amino acid residues in conserved exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn210-Xaa-Xaa-Xaa-Phe-Asp), and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification fidelity (error rate) of the DNA polymerases were altered by mutagenesis. However, long and accurate PCR by a single-type of mutant DNA polymerase was very difficult. The wild-type DNA polymerase (WT) and its exonuclease deficient mutant (N210D) were mixed in different ratio and their characteristics in PCR were examined. When the mixed enzyme (WT and N210D) was made at the ratio of 1:40, long PCR (15 kbp) at lower mutation frequency could be efficiently achieved.     

Promotion of hematopoietic differentiation from mouse induced pluripotent stem cells by transient HoxB4 transduction

Ectopic expression of HoxB4 in embryonic stem (ES) cells leads to an efficient production of hematopoietic cells, including hematopoietic stem/progenitor cells. Previous studies have utilized a constitutive HoxB4 expression system or tetracycline-regulated HoxB4 expression system to induce hematopoietic cells from ES cells. However, these methods cannot be applied therapeutically due to the risk of transgenes being integrated into the host genome. Here, we report the promotion of hematopoietic differentiation from mouse ES cells and induced pluripotent stem (iPS) cells by transient HoxB4 expression using an adenovirus (Ad) vector. Ad vector could mediate efficient HoxB4 expression in ES cell-derived embryoid bodies (ES-EBs) and iPS-EBs, and its expression was decreased during cultivation, showing that Ad vector transduction was transient. A colony-forming assay revealed that the number of hematopoietic progenitor cells with colony-forming potential in HoxB4-transduced cells was significantly increased in comparison with that in non-transduced cells or LacZ-transduced cells. HoxB4-transduced cells also showed more efficient generation of CD41-, CD45-, or Sca-1-positive cells than control cells. These results indicate that transient, but not constitutive, HoxB4 expression is sufficient to augment the hematopoietic differentiation of ES and iPS cells, and that our method would be useful for clinical applications, such as cell transplantation therapy.     

Identification of growth-promoting factors from various glioma cell lines and partial purification of the factor from C6 solid tumor

Growth-promoting factors in the extracts of various glioma cell lines (C6, LRM55 and 354A) were investigated. The cell extracts of astrocytoma (C6) and mixed glioma (LRM55) showed a high mitogenic activity to normal glioblasts. With its low content of intracellular growth-promoting factor, rat peripheral glioma (354A) exhibited a high proliferative response to C6 cell extracts. The factor which was partially purified from C6 solid tumor by ion exchange and gel filtration column chromatographies had two forms of different molecular weights (150,000 Mr and 35,000 Mr) and the low molecular weight form was further split into two acidic proteins (pl 5.0 and pl 6.0) by isoelectric focusing. The mitogenic activity of the factor was susceptible to heat and to proteases, and the factor showed no esteropeptidase activity. These physicochemical properties closely resemble those of glia maturation factor from porcine brains.     

Coordinated Behaviour in Pigeon Flocks

We analysed pigeon flock flights using GPS trajectory data to reveal the most important kinematic aspects of flocking behaviour. We quantitatively investigated the internal motion of the flock based on pairwise statistics and found the following general relationships in all datasets: i) the temporal order of decisions characterised by the delay between directional changes is strictly related to the spatial order characterised by the longitudinal relative position within the flock; ii) during circling motion, pigeons use a mixture of two idealised and fundamentally different turning strategies, namely, parallel-path and equal-radius type turning. While pigeons tend to maintain their relative position within the flock on average, as in the parallel-path approximation, those who turn later also get behind as in the equal-radius case. Equal-radius type turning also tends to be expressed more during smaller radius turns.     

Chemical Profiling of Jatropha Tissues under Different Torrefaction Conditions: Application to Biomass Waste Recovery

Gradual depletion of the world petroleum reserves and the impact of environmental pollution highlight the importance of developing alternative energy resources such as plant biomass. To address these issues, intensive research has focused on the plant Jatropha curcas, which serves as a rich source of biodiesel because of its high seed oil content. However, producing biodiesel from Jatropha generates large amounts of biomass waste that are difficult to use. Therefore, the objective of our research was to analyze the effects of different conditions of torrefaction on Jatropha biomass. Six different types of Jatropha tissues (seed coat, kernel, stem, xylem, bark, and leaf) were torrefied at four different temperature conditions (200°C, 250°C, 300°C, and 350°C), and changes in the metabolite composition of the torrefied products were determined by Fourier transform-infrared spectroscopy and nuclear magnetic resonance analyses. Cellulose was gradually converted to oligosaccharides in the temperature range of 200°C–300°C and completely degraded at 350°C. Hemicellulose residues showed different degradation patterns depending on the tissue, whereas glucuronoxylan efficiently decomposed between 300°C and 350°C. Heat-induced depolymerization of starch to maltodextrin started between 200°C and 250°C, and oligomer sugar structure degradation occurred at higher temperatures. Lignin degraded at each temperature, e.g., syringyl (S) degraded at lower temperatures than guaiacyl (G). Finally, the toxic compound phorbol ester degraded gradually starting at 235°C and efficiently just below 300°C. These results suggest that torrefaction is a feasible treatment for further processing of residual biomass to biorefinery stock or fertilizer.     

Capturing microscopic features of bone remodeling into a macroscopic model based on biological rationales of bone adaptation

To understand Wolff’s law, bone adaptation by remodeling at the cellular and tissue levels has been discussed extensively through experimental and simulation studies. For the clinical application of a bone remodeling simulation, it is significant to establish a macroscopic model that incorporates clarified microscopic mechanisms. In this study, we proposed novel macroscopic models based on the microscopic mechanism of osteocytic mechanosensing, in which the flow of fluid in the lacuno-canalicular porosity generated by fluid pressure gradients plays an important role, and theoretically evaluated the proposed models, taking biological rationales of bone adaptation into account. The proposed models were categorized into two groups according to whether the remodeling equilibrium state was defined globally or locally, i.e., the global or local uniformity models. Each remodeling stimulus in the proposed models was quantitatively evaluated through image-based finite element analyses of a swine cancellous bone, according to two introduced criteria associated with the trabecular volume and orientation at remodeling equilibrium based on biological rationales. The evaluation suggested that nonuniformity of the mean stress gradient in the local uniformity model, one of the proposed stimuli, has high validity. Furthermore, the adaptive potential of each stimulus was discussed based on spatial distribution of a remodeling stimulus on the trabecular surface. The theoretical consideration of a remodeling stimulus based on biological rationales of bone adaptation would contribute to the establishment of a clinically applicable and reliable simulation model of bone remodeling.     

Histamine N-methyltransferase from rat kidney. Cloning, nucleotide sequence, and expression in Escherichia coli cells.

Complementary DNA clones encoding rat kidney histamine N-methyltransferase have been isolated using synthetic oligonucleotide probes based on partial amino acid sequences of tryptic peptides of the purified enzyme. The 1.3-kilobase cDNA consisted of a 5'-noncoding region of 8 nucleotides, a coding region of 885 nucleotides, and a 3'-noncoding region of 369 nucleotides. The encoded protein of 295 amino acid residues had a calculated molecular weight of 33,940.2. After introduction of a prokaryotic expression vector containing the isolated cDNA, Escherichia coli cells expressed histamine N-methyltransferase activity. The enzyme expressed in these cells was isolated and purified as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whose mobility was identical to the natural enzyme purified from rat kidney. The recombinant enzyme had Vmax and Km values for both histamine and S-adenosylmethionine identical to those of the natural enzyme. All of the inhibitors of the natural enzyme tested showed similar Ki values on both recombinant and natural enzyme.