A new fibrous protein from Tetrahymena pyriformis

Isolation and characterization of a fibrous protein component which might be associated with contractile ring of a dividing Tetrahymena cell were attempted by making use of coprecipitation of the protein with rabbit skeletal muscle myosin. The protein was purified to homogeneity by ammonium sulfate fractionation between ...Thesis--University of Tsukuba, D.Sc.(B), no. 32, 1980. 7. 3

Roles of Three Domains of Tetrahymena eEF1Ain Bundling F-actin

The conventional role of eukaryotic elongation factor 1A (eEF1A) is to transport aminoacyl tRNA to the A site of ribosomes during the peptide elongation phase of protein synthesis. eEF1A also is involved in regulating the dynamics of microtubules and actin filaments in cytoplasm. In Tetrahymena, eEF1A forms homodimers and bundles F-actin. Ca2+/calmodulin (CaM) causes reversion of the eEF1A dimer to the monomer, which loosens F-actin bundling, and then Ca2+/CaM/eEF1A monomer complexes dissociate from F-actin. eEF1A consists of three domains in all eukaryotic species, but the individual roles of the Tetrahymena eEF1A domains in bundling F-actin are unknown. In this study, we investigated the interaction of each domain with F-actin, recombinant Tetrahymena CaM, and eEF1A itself in vitro, using three glutathione-S-transferase-domain fusion proteins (GST-dm1, -2, and -3). We found that only GST-dm3 bound to F-actin and influences dimer formation, but that all three domains bound to Tetrahymena CaM in a Ca2+-dependent manner. The critical Ca2+ concentration for binding among three domains of eEF1A and CaM were ≤100 nM for domain 1, 100 nM to 1 μM for domain 3, and >1 μM for domain 2, whereas stimulation of and subsequent Ca2+ influx through Ca2+ channels raise the cellular Ca2+ concentration from the basal level of ~100 nM to ~10 μM, suggesting that domain 3 has a pivotal role in Ca2+/CaM regulation of eEF1A

Tetrahymena Elongation Factor-1α Binds to Hsp70 Family Proteins

Translation elongation factor 1α (EF-1α) catalyzes the GTP-dependent binding of aminoacyl-tRNA to the ribosome. We previously reported that Tetrahymena EF-1α induced bundles of rabbit skeletalmuscle F-actin as well as Tetrahymena F-actin (Kurasawa et al., (1996) Zool. Sci. 13: 371–375), andthat Ca2+/calmodulin (CaM) regulated the F-actin-bundling activity of EF-1α without inhibition of the bindingbetween EF-1α and F-actin (Kurasawa et al.,(1996) J. Biochem. 119: 791–798). In this study, we investigatedEF-1α-binding proteins in Tetrahymena using a Tetrahymena EF-1α affinity column. TetrahymenaEF-1α bound directly to 74 kDa, 77 kDa, and 78 kDa proteins, in addition to CaM. The bindings of 74 kDa, 77kDa, and 78 kDa proteins to Tetrahymena EF-1α were Ca2+-independent and ATP-sensitive. The N-terminalamino acid sequence of the 74 kDa protein was similar to those of 70 kDa heat shock protein (hsp70) family

Distorted antibody repertoire developed in the absence of pre-B cell receptor formation

The pre-B cell receptor (pre-BCR), consisting of the μ heavy chain (μHC) and the surrogate light chain (SLC, Vpre-B and λ5), plays important roles during B cell development. The formation of the pre-BCR, which enables the nascent immunoglobulin HC to associate with the SLC, is considered a prerequisite for B cell development. However, a significant number of peripheral mature (leaky) B cells exist in SLC-deficient mice. These leaky B cells develop in the absence of pre-BCR and do not undergo the pre-BCR checkpoint. The antibody repertoires of leaky B cells thus reflect the absence of pre-BCR function. To investigate how the absence of the pre-BCR is circumvented by these leaky-B cells and examine the effect of the pre-BCR checkpoint on the antibody system, we analyzed the antibody repertoires of λ5-deficient (λ5−/−) mice using next-generation sequencing. In λ5−/− mice, spleen B cells displayed different patterns of VDJ-usage, relative to those in wild-type (WT) mice. Moreover, leaky B cells were neither derived from unusual B2 cells, characterized by particular LC gene rearrangements in the absence of pre-BCR signaling, nor from B1 cells, originating from different B cell progenitors. Analysis of the CDR-H3 amino acid sequences of μ-chain repertoires revealed that certain bone marrow B cells with particular CDR-H3 profiles undergo clonal expansion in λ5−/− mice. Part of these CDR-H3s contain arginine(s) in the middle of the CDR-H3 loop in λ5−/− mice, whereas few arginine(s) exist in this middle loop in WT CDR-H3s in the absence of clonal expansion. This CDR-H3 feature in λ5−/− mice presumably reflects the role of the pre-BCR in autoantibody regulation, since arginine(s) are often found in the antigen-binding site of autoantibodies. Here, we present a unique viewpoint on the role of pre-BCR, by assessing the whole antibody repertoire formed in SLC-deficient mice

Black Tea High-Molecular-Weight Polyphenol Stimulates Exercise Training-Induced Improvement of Endurance Capacity in Mouse via the Link between AMPK and GLUT4

Aerobic exercise can promote “fast-to-slow transition” in skeletal muscles, i.e. an increase in oxidative fibers, mitochondria, and myoglobin and improvement in glucose and lipid metabolism. Here, we found that mice administered Mitochondria Activation Factor (MAF) combined with exercise training could run longer distances and for a longer time compared with the exercise only group; MAF is a high-molecular-weight polyphenol purified from black tea. Furthermore, MAF intake combined with exercise training increased phosphorylation of AMPK and mRNA level of glucose transporter 4 (GLUT4). Thus, our data demonstrate for the first time that MAF activates exercise training-induced intracellular signaling pathways that involve AMPK, and improves endurance capacity

High-Molecular-Weight Polyphenol-Rich Fraction of Black Tea Does Not Prevent Atrophy by Unloading, But Promotes Soleus Muscle Mass Recovery from Atrophy in Mice

Previously, we reported that polyphenol-rich fraction (named E80) promotes skeletal muscle hypertrophy induced by functional overload in mice. This study indicates that E80 has potential for affecting skeletal muscle mass. Then, we evaluate the effect of E80 on atrophic and recovery conditions of skeletal muscle in mice. Hindlimb suspension (unloading) and relanding (reloading) are used extensively to observe disuse muscle atrophy and subsequent muscle mass recovery from atrophy. Eight-week old C57BL/6 mice were fed either a normal diet or a diet containing 0.5% E80 for two weeks under conditions of hindlimb suspension and a subsequent 5 or 10 days of reloading. We found that E80 administration did not prevent atrophy during hindlimb suspension, but promoted recovery of slow-twitch (soleus) muscle mass from atrophy induced by hindlimb suspension. After five days of reloading, we discovered that phosphorylation of the Akt/mammalian target of rapamycin (mTOR) pathway proteins, such as Akt and P70 ribosomal protein S6 kinase (S6K), was activated in the muscle. Therefore, E80 administration accelerated mTOR signal and increased protein synthesis in the reloaded soleus muscle

Coincidence analysis to search for inspiraling compact binaries using TAMA300 and LISM data

Japanese laser interferometric gravitational wave detectors, TAMA300 and LISM, performed a coincident observation during 2001. We perform a coincidence analysis to search for inspiraling compact binaries. The length of data used for the coincidence analysis is 275 hours when both TAMA300 and LISM detectors are operated simultaneously. TAMA300 and LISM data are analyzed by matched filtering, and candidates for gravitational wave events are obtained. If there is a true gravitational wave signal, it should appear in both data of detectors with consistent waveforms characterized by masses of stars, amplitude of the signal, the coalescence time and so on. We introduce a set of coincidence conditions of the parameters, and search for coincident events. This procedure reduces the number of fake events considerably, by a factor $\sim 10^{-4}$ compared with the number of fake events in single detector analysis. We find that the number of events after imposing the coincidence conditions is consistent with the number of accidental coincidences produced purely by noise. We thus find no evidence of gravitational wave signals. We obtain an upper limit of 0.046 /hours (CL $= 90$) to the Galactic event rate within 1kpc from the Earth. The method used in this paper can be applied straightforwardly to the case of coincidence observations with more than two detectors with arbitrary arm directions.Comment: 28 pages, 17 figures, Replaced with the version to be published in Physical Review

Results of the search for inspiraling compact star binaries from TAMA300's observation in 2000-2004

We analyze the data of TAMA300 detector to search for gravitational waves from inspiraling compact star binaries with masses of the component stars in the range 1-3Msolar. In this analysis, 2705 hours of data, taken during the years 2000-2004, are used for the event search. We combine the results of different observation runs, and obtained a single upper limit on the rate of the coalescence of compact binaries in our Galaxy of 20 per year at a 90% confidence level. In this upper limit, the effect of various systematic errors such like the uncertainty of the background estimation and the calibration of the detector's sensitivity are included.Comment: 8 pages, 4 Postscript figures, uses revtex4.sty The author list was correcte

DECIGO pathfinder

DECIGO pathfinder (DPF) is a milestone satellite mission for DECIGO (DECi-hertz Interferometer Gravitational wave Observatory) which is a future space gravitational wave antenna. DECIGO is expected to provide us fruitful insights into the universe, in particular about dark energy, a formation mechanism of supermassive black holes, and the inflation of the universe. Since DECIGO will be an extremely large mission which will formed by three drag-free spacecraft with 1000m separation, it is significant to gain the technical feasibility of DECIGO before its planned launch in 2024. Thus, we are planning to launch two milestone missions: DPF and pre-DECIGO. The conceptual design and current status of the first milestone mission, DPF, are reviewed in this article

Molecular dissection of the actin-binding ability of the fission yeast α-actinin, Ain1, in vitro and in vivo

A contractile ring (CR) is involved in cytokinesis in animal and yeast cells. Although several types of actin-bundling proteins associate with F-actin in the CR, their individual roles in the CR have not yet been elucidated in detail. Ain1 is the sole α-actinin homologue in the fission yeast Schizosaccharomyces pombe and specifically localizes to the CR with a high turnover rate. S. pombe cells lacking the ain1+ gene show defects in cytokinesis under stress conditions. We herein investigated the biochemical activity and cellular localization mechanisms of Ain1. Ain1 showed weaker affinity to F-actin in vitro than other actin-bundling proteins in S. pombe. We identified a mutation that presumably loosened the interaction between two calponin-homology domains constituting the single actin-binding domain (ABD) of Ain1, which strengthened the actin-binding activity of Ain1. This mutant protein induced a deformation in the ring shape of the CR. Neither a truncated protein consisting only of an N-terminal ABD nor a truncated protein lacking a C-terminal region containing an EF-hand motif localized to the CR, whereas the latter was involved in the bundling of F-actin in vitro. We herein propose detailed mechanisms for how each part of the molecule is involved in the proper cellular localization and function of Ain1