Immuno‐detection of mRNA‐binding protein complex in human cells under transmission electron microscopy

Transit from the nuclear complex to the cytoplasm through the nuclear pore complex permits modification of mRNA, including processing such as splicing, capping, and polyadenylation, etc. At each of these events, mRNA interacts with various proteins to form mRNA‐protein complex. Visualizing the mRNA is crucial for understanding the mechanisms underlying mRNA processing and elucidating its structure and recent advances in mRNA imaging allow detection of real‐time mRNA localization in living cells. However, these techniques revealed only the location of mRNA but cannot visualize the conformation of mRNA‐protein complex in cells. On the other hand, transmission electron microscopy has been used to visualize the structure of the Balbiani ring‐derived large mRNA, but their observations were limited to the insect cells. In this study, we visualized the structure of mRNA‐protein complex in human culture cells by using immuno‐electron microscopy. Through immuno‐detection, an mRNA exon junction binding complex Y14, and its binding protien Upf2, different gold particle patterns were imaged with transmission electron microscopy and analyzed. Characteristic linear and stacked particle orientation were observed. Across the nuclear membrane, only linear aggregation pattern was observed, whereas the stacked aggregation pattern was detected in the cytoplasm. Our method is able to visualize mRNA‐conformation and applicable to many cell types, including mammalian cells, where genes can easily be manipulated.     

The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.     

Remodeling of Bone Marrow Niches and Roles of Exosomes in Leukemia

Leukemia is a hematological malignancy that originates from hematopoietic stem cells in the bone marrow. Significant progress has made in understanding its pathogensis and in establishing chemotherapy and hematopoietic stem cell transplantation therapy (HSCT). However, while the successive development of new therapies, such as molecular-targeted therapy and immunotherapy, have resulted in remarkable advances, the fact remains that some patients still cannot be saved, and resistance to treatment and relapse are still problems that need to be solved in leukemia patients. The bone marrow (BM) niche is a microenvironment that includes hematopoietic stem cells and their supporting cells. Leukemia cells interact with bone marrow niches and modulate them, not only inducing molecular and functional changes but also switching to niches favored by leukemia cells. The latter are closely associated with leukemia progression, suppression of normal hematopoiesis, and chemotherapy resistance, which is precisely the area of ongoing study. Exosomes play an important role in cell-to-cell communication, not only with cells in close proximity but also with those more distant due to the nature of exosomal circulation via body fluids. In leukemia, exosomes play important roles in leukemogenesis, disease progression, and organ invasion, and their usefulness in the diagnosis and treatment of leukemia has recently been reported. The interaction between leukemia cell-derived exosomes and the BM microenvironment has received particular attention. Their interaction is believed to play a very important role; in addition to their diagnostic value, exosomes could serve as a marker for monitoring treatment efficacy and as an aid in overcoming drug resistance, among the many problems in leukemia patients that have yet to be overcome. In this paper, we will review bone marrow niches in leukemia, findings on leukemia-derived exosomes, and exosome-induced changes in bone marrow niches.     

Quantitative characterization of microcracking in A533B steel by acoustic emission

The initiation of microcracks at MsS inclusions and their coalescence during fracture toughness tests of ASTM A533B steel compact tension specimens were detected by an acoustic emission technique. The sources of emission, which are characterized by the crack size and orientation, fracture mode, and time history, are represented by a time-dependent moment tensor. Since the recorded waveforms are related to the moment tensor, the Green’s displacement functions of the specimen, and the transfer function of the recording system by a convolution integral in the theory of elastodynamics, the moment tensors are determined by techniques of deconvolution. Two types of sources were found: a tensile mode of crack caused by decohesion of MnS inclusion and the coalescence of microcracks with mixed tensile and shear modes. The dimensions and time durations of microcracks were also estimated in this study. T{upakanori} O{uphira}, Late Associate Professor of the Department of Metallurgy and Materials Science, University of Tokyo, Tokyo, Japan, died on December 9, 1987, at the age of 33 years. He had been a Visiting Scientist at Cornell University from 1984 through 1986 under the sponsorship of the Japan Society for the promotion of Science.     

Artificial surfactant (Surfactant TA) modulates adherence and superoxide production of neutrophils

Neutrophils cause lung injuries by releasing proteases and active oxygen radicals in patients with acute respiratory distress syndrome (ARDS). Artificial surfactant is used to replace native surfactant whose functions are deteriorated by serum-derived inhibitors in these patients. We investigated potential interactions between exogenous surfactant (Surfactant TA) and neutrophils in in vivo and in vitro experimental models. Neutrophil alveolitis was induced in hamster lungs by the intratracheal administration of bleomycin (5 mg/kg) on Day 0. Some of the animals were followed by replacement with Surfactant TA (5 and 10 mg/100 g body weight) on Day 1. Alveolar cells were harvested by lung lavage on Day 2. The numbers of the neutrophils obtained from the lungs treated with bleomycin and Surfactant TA were unchanged, but the superoxide production from these cells was significantly decreased when compared with control animals (no Surfactant TA). From the in vitro experiments, Surfactant TA was shown to inhibit adherence and superoxide production of human neutrophils. These effects were derived from the heat-resistant components of Surfactant TA and were mimicked by treatment with liposomes of dipalmitoyl phosphatidylcholine. Surfactant-TA-treated neutrophils were demonstrated to have picnotic nuclei and to express Fas antigens, which were characteristic of apoptotic cells. These results suggest that exogenous Surfactant TA may play an important role not only in improving surfactant functions but in preventing neutrophils from further activation, probably through enhancing apoptosis.     

Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface

We studied human immunodeficiency virus type 1 (HIV-1) Nef protein biochemically and histologically. HIV-1 Nef, derived from baculosystem and from cells infected with HIV-1, formed homomeric monomers, dimers, trimers, and further polymers. These oligomers were non-covalently associated. In cells infected with HIV-1, Nef molecules were clustered at the cell surface as well as cytoplasm. Our previous results have indicated that the Nef on the surface of cells infected with HIV-1 is cytotoxic against uninfected CD4+ T cells. Thus, it is very likely that the HIV-1-mediated cytotoxic reaction is due, at least in part, to the clustered localization of oligomeric Nef on the cell surface.     

Comparisons between silk fibroin nonwoven electrospun fabrics using aqueous and formic acid solutions

The objective of this study is to compare the spinnability, morphology, structure, mechanical properties, and cell compatibility of the silk fibroin nanofiber nonwoven electrospun fabrics using aqueous (AQ) solution and formic acid (FA) solution. The lower limit concentration was 5?wt% and 3?wt% of AQ solution and FA solution for electrospinning, respectively. The fiber diameter of electrospun fabric using FA solution was larger than that using aqueous solution at the same concentration. The secondary structure contents of silk fabrics were same between AQ and FA solutions. FA was remained in silk nanofibers, and the remained FA could be neutralized. Young’s modulus and cell adhesion on electrospun fabric using FA was lower than that using AQ solution. On the contrary, lower cell proliferation rate on electrospun fabric using FA was kept even after neutralization.     

Spontaneous behavior for cooperation through interaction: An emotionally intelligent robot system

This paper deals withspontaneous behavior for cooperation through interaction in a distributed autonomous robot system. Though a human gives the robots evaluation functions for the relation of cooperation among robots, each robot decides its behavior depending on its environment, its experience, and the behavior of other robots. The robot acquires a model of the behavior of the other robots through learning. Inspired by biological systems, the robot's behaviors are interpreted as emotional by an observer of the system. In psychology, the emotions have been considered to play important roles for generation of motivation and behavior selection. In this paper, the robot's behaviors are interpreted as follows: each robot feels frustration when its behavior decision does not fit its environment. Then, it changes its behavior to change its situation actively and spontaneously. The results show potential of intelligent behavior by emotions. This work was presented, in part, at the International Symposium on Artificial Life and Robotics, Oita, Japan, February 18–20, 1996     

Optimal design method with biomechanical analysis for a work environment reducing physical workload: illustration by application to work table height design

A work environment should be designed to minimise physical workload. We propose an optimal work environment design method to accomplish this, in which joint moment ratios were calculated by biomechanical analyses through digital human modelling (DHM) and were used to indicate physical workload. The work environment design problem was formulated as a multi-objective optimisation problem, minimising the average and maximum joint moment ratio values to determine the optimal work environment. Sequential approximate optimisation, which improves the accuracy of the response surface by sequentially adding new sampling points from a simulation, was applied to efficiently obtain a precise optimal solution. The proposed method was applied to designing a work table height for light assembly tasks. This method determined the optimal work table height considering the anthropometric diversity of workers from a relatively small number of subjects. Through this case study, the validity of the proposed method is discussed.     

Synthesis and Application of Stable Copper Oxide Nanoparticle Suspensions for Nanoparticulate Film Fabrication

Colloidal suspensions of copper oxide (CuO) nanoparticles were prepared by an alcothermal method, in which copper acetate was reacted with sodium hydroxide in the presence of acetic acid in ethanol at 78°C. The prepared suspension was stable for up to 1 month without stabilizers such as surfactants. Transmission electron microscopy analyses revealed that the suspension contained nanosized CuO particles of 5–10 nm size with a narrow size distribution. Nanoparticulate CuO films packed with grains smaller than 60 nm were fabricated on Si substrates by spin coating a suspension of CuO nanoparticles and subsequent heat treatment at 500°C.