N-terminal Ago-binding domain of GW182 contains a tryptophan-rich region that confer binding to the CCR4-NOT complex.

GW182 family proteins are a key component of microRNA-protein complex eliciting translational repression and/or degradation of microRNA-targets. The microRNAs in complex with Argonaute proteins bind to target mRNAs, and GW182 proteins are recruited by association with Argonaute proteins. The GW182 protein acts as a scaffold that links the Argonaute protein to silencing machineries including the CCR4-NOT complex which accelerates deadenylation and inhibits translation. The carboxyl-terminal effector domain of GW182 protein, also called the silencing domain, has been shown to bind to the subunits of the CCR4-NOT complex, the CNOT1 and the CNOT9. Here we show that a small region within the amino-terminal Argonaute-binding domain of human GW182/TNRC6A can associate with the CCR4-NOT complex. This region resides between the two Argonaute-binding sites and contains reiterated GW/WG-motifs. Alanine mutation experiments showed that multiple tryptophan residues are required for the association with the CCR4-NOT complex. Furthermore, co-expression and immunoprecipitation assays suggested that the CNOT9 subunit of the CCR4-NOT complex is a possible binding partner of this region. Our work, taken together with previous studies, indicates that the human GW182 protein contains multiple binding interfaces to the CCR4-NOT complex.

Comparison of timing and force control of foot tapping between elderly and young subjects.

[Purpose] To examine the ability of young and elderly individuals to control the timing and force of periodic sequential foot tapping. [Subjects and Methods] Participants were 10 young (age, 22.1 ± 4.3 years) and 10 elderly individuals (74.8 ± 6.7 years) who were healthy and active. The foot tapping task consisted of practice (stimulus-synchronized tapping with visual feedback) and recall trials (self-paced tapping without visual feedback), periodically performed in this order, at 500-, 1,000-, and 2,000-ms target interstimulus-onset intervals, with a target force of 20% maximum voluntary contraction of the ankle plantar-flexor muscle. [Results] The coefficients of variation of force and intertap interval, used for quantifying the steadiness of the trials, were significantly greater in the elderly than in the young individuals. At the 500-ms interstimulus-onset interval, age-related effects were observed on the normalized mean absolute error of force, which was used to quantify the accuracy of the trials. The coefficients of variation of intertap interval for elderly individuals were significantly greater in the practice than in the recall trials at the 500- and 1,000-ms interstimulus-onset intervals. [Conclusion] The elderly individuals exhibited greater force and timing variability than the young individuals and showed impaired visuomotor processing during foot tapping sequences.

QSAR analysis of substituent effects on tambjamine anion transporters.

The transmembrane anion transport activity of 43 synthetic molecules based on the structure of marine alkaloid tambjamine were assessed in model phospholipid (POPC) liposomes. The anionophoric activity of these molecules showed a parabolic dependence with lipophilicity, with an optimum range for transport efficiency. Using a quantitative structure-transport activity (QSAR) approach it was possible to rationalize these results and to quantify the contribution of lipophilicity to the transport activity of these derivatives. While the optimal value of log P and the curvature of the parabolic dependence is a property of the membrane (and so similar for the different series of substituents) we found that for relatively simple substituents in certain locations on the tambjamine core, hydrophobic interactions clearly dominate, but for others, more specific interactions are present that change the position of the membrane hydrophobicity parabolic envelope.

Bent molecules with a 60° central core angle that form B7 and B2 phases.

Small-angle bent-core liquid-crystalline (LC) molecules based on a 1,2-bis(phenylethynyl)benzene central core have been synthesized that form banana smectic phases with a ferroelectric B7-antiferroelectric B2 phase sequence upon cooling. The formation of polar, switchable ferro-/antiferroelectric banana phases indicates that, despite the low core bend angle of approximately 60°, banana smectic phases are still formed with the bend direction parallel to the layer. This study offers significant evidence that shows bent-core molecules with a 60° bend angle can form the well-known B2 and B7 banana phases. Consequently, it may lead to the preparation of a wide variety of novel bent molecules with low bend angles that spontaneously form an LC phase with both polarization and chirality.

Structural analysis of a banana-liquid crystal in the B4 phase by solid-state NMR.

In this paper, we present a structural investigation of 1,3-phenylene bis[4-((4-10-decyloxyphenyl)iminomethyl)-benzoate], known as a banana-liquid crystal, in the B4 phase, which was performed by solid-state nuclear magnetic resonance (NMR) methodology combined with quantum chemical calculations. The present solid-state NMR measurements including (13)C CPMAS, 2D TOSS-deTOSS, dipole-dephase, 1D and 2D EXSY, and MAS-j-HMQC provided accurate spectral assignments and unambiguous NMR parameters such as (13)C chemical shift tensors, which were used for construction of the three-dimensional structure with the aid of density functional theory calculations. In the obtained molecular structure, two arms of the bent-core molecule are asymmetrically expanded such that the direction of the dipole moment is off alignment with respect to the middle line of the center benzene ring. It is this antisymmetric structure that is the origin of the twisted helical system in the B4 phase.

Tethering of proteins to RNAs using the bovine immunodeficiency virus-Tat peptide and BIV-TAR RNA.

To study the functions of RNA-binding proteins independent of their RNA-binding activity, tethering methods have been developed, based on the use of the RNA-binding domain of a well-characterized RNA-binding protein and its target RNA. Two bacteriophage proteins have mainly been used as tethers: the MS2 coat protein and the lambda N protein. Here we report an alternative system using the Tat (trans-activator) peptide from the bovine immunodeficiency virus (BIV), which binds to BIV-TAR (trans-activation response) RNA. We demonstrate the usefulness of this system by applying it to the analysis of the TNRC6B protein, a component of the microRNA-induced silencing complex.

Characterization of the nuclear- and plastid-encoded secA-homologous genes in the unicellular red alga Cyanidioschyzon merolae.

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.

Interaction interference between arm and leg: division of attention through muscle force regulation.

The first purpose of this study was to examine whether decreases in muscle force similar to the bilateral deficit occur during simultaneous use of arm and leg. The second purpose was to examine the effect on the muscle force of one leg by a division of attention through the regulation of the muscle force in the arm. Six participants completed each of the following three tasks in a random order: (1) maximal unilateral flexion of the right or left elbow, (2) maximal unilateral extension of the left knee, and (3) multilimb effort (a maximal contraction of the muscles in the leg while maintaining a constant submaximal isometric elbow flexion force at 25%, 50%, 75%, or 100% MVC). The results showed that muscle force was lower during simultaneous exertion of arm and leg than during exertion of one limb alone. The maximal knee extension force was significantly (p<.05) lower, by as much as 40% or so, during regulation at 25% MVC. The division of attention is also thought to be involved in task execution and may thus explain the test results. A decrease in the muscle force of the leg due to the level of regulation of the muscle force of the arm indicates that the regulation of the muscle force affects the division of attention, and the finer level of muscle force regulation is a task that requires greater attention. When the muscle force is precisely controlled, a more accurate and more appropriate adjustment is required to focus attention.

Mammalian GW182 contains multiple Argonaute-binding sites and functions in microRNA-mediated translational repression.

In mammalian cells, microRNAs (miRNAs) are incorporated into miRNA-induced silencing complexes (miRISCs), which regulate protein expression post-transcriptionally through binding to 3'-untranslated regions of target mRNAs. Argonaute2 (Ago2), a key component of the miRISC, recruits GW182, a component of the processing body (GW/P-body), to the target mRNAs. To elucidate the function of GW182 in an miRNA-mediated translational repression, we analyzed Argonaute-binding sites in GW182. We found that human GW182 contains three binding sites for Ago2, within the amino-terminal glycine tryptophan (GW/WG)-repeated region that is characteristic of the GW182 family proteins. We also found that the first and second Ago2-binding site is conserved within the amino-terminal half of TNRC6B, which is a paralog of GW182. Each of the Ago-binding sites is alone sufficient to bind Ago2. Furthermore, we demonstrated that multiple Argonaute proteins were connected via the GW182 protein. A GW182 fragment containing the Ago2-binding region partially relieved let-7-mediated repression of protein synthesis in a mammalian cell-free system. Coincidentally, let-7-directed target mRNA deadenylation was delayed. Together, these results strongly suggested that the interactions of GW182 with Argonautes may induce the formation of large complexes containing miRNA target mRNAs, and may be critical for miRNA-mediated translational repression.

A new simple performance test focused on agility in elderly people: The Ten Step Test.

BACKGROUND: Not only the reduction of muscle strength or balance, but also the reduction of the agility are regarded as important factors of falls in elderly people. If an agility test for elderly people is established, the precision of the fall prediction rises and can be used for individual training. OBJECTIVES: To develop a new performance test focused on agility for elderly people and to evaluate the usefulness of this test. DESIGN: Cross-sectional study. SETTING: The Welfare Center of Kagami Town, Kagami Town Office, etc., Kochi, Japan. PARTICIPANTS: 828 community-dwelling, independent adults aged 20-99 years with no obvious cognitive or functional disability, were randomly recruited from Kagami town and the surrounding areas. MEASUREMENTS: The Ten Step Test (TST, a new performance test), motor reaction time (MRT), knee extensor isometric strength, single leg standing time (SLST), and some other tests were used to evaluate the criterion-related validity and the content validity. TST was developed as a modified version of other step tests which require the subjects to place the whole foot on a block, then return it to the floor. In addition, female participants over 70 were asked whether or not they had fallen in the past year. RESULTS: Excellent reliability for TST was found for interrelation (intra-class correlation coefficients, ICC = 0.96), and re-test reliability was sufficient (ICC = 0.86). Evidence for criterion-related validity was found through high single correlation with the timed supine-to-stand (r = 0.68) and high single correlation with MRT (r = 0.59). In addition, content validity was found through low correlation with knee extensor strength (-0.35) and SLST (-0.36) in 112 women over 70 years of age. The error rate by TST to predict falls (35.2%) was lower than the error rate by muscle strength (44.4%) and the balance (38.7%). TST confirmed decline after 50 years of age, and it conformed to a cubic curve. CONCLUSION: The findings indicate that TST is a reliable measure of agility, and it can help to predict the risk of falls. The decline of agility accelerates after 50 years of age. It shows that the decline of agility differs from the decline of leg muscle strength and balance.

Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system.

MicroRNAs (miRNAs) are incorporated into miRNP complexes and regulate protein expression post-transcriptionally through binding to 3'-untranslated regions of target mRNAs. Here we describe a recapitulation of let-7 miRNA-mediated translational repression in a cell-free system, which was established with extracts prepared from HEK293F cells overexpressing miRNA pathway components. In this system, both the cap and poly(A) tail are required for the translational repression, and let-7 directs the deadenylation of target mRNAs. Our work suggests that let-7 miRNPs containing Argonaute and GW182 impair the synergistic enhancement of translation by the 5'-cap and 3'-poly(A) tail, resulting in translational repression.