Early development and neurogenesis of Temnopleurus reevesii

Sea urchins are model non‐chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15–30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology.     

Antibacterial activity of disodium succinoyl glycyrrhetinate,a derivative of glycyrrhetinic acid against Streptococcus mutans

Streptococcus mutans is a cariogenic bacterium that localizes in the oral cavity. Glycyrrhetinic acid (GRA) is a major component of licorice extract. GRA and several derivatives, including disodium succinoyl glycyrrhetinate (GR‐SU), are known to have anti‐inflammatory effects in humans. In this study, the antimicrobial effect of GRA and its derivatives against the S. mutans UA159 strain were investigated. Minimum inhibitory concentrations (MICs) of GRA and GR‐SU showed antibacterial activity against the S. mutans strain, whereas other tested derivatives did not. Because GR‐SU is more soluble than GRA, GR‐SU was used for further experiments. The antibacterial activity of GR‐SU against 100 S. mutans strains was evaluated and it was found that all strains are susceptible to GR‐SU, with MIC values below 256 µg/mL. A cell viability assay showed that GR‐SU has a bacteriostatic effect on S. mutans cells. As to growth kinetics, sub‐MICs of GR‐SU inhibited growth. The effect of GR‐SU on S. mutans virulence was then investigated. GR‐SU at sub‐MICs suppresses biofilm formation. Additionally, GR‐SU greatly suppresses the pH drop caused by the addition of glucose and glucose‐induced expression of the genes responsible for acid production (ldh and pykF) and tolerance (aguD and atpD). Additionally, expression of enolase, which is responsible for the carbohydrate phosphotransferase system, was not increased in the presence of GR‐SU, indicating that GR‐SU suppresses incorporation of sugars into S. mutans. In conclusion, GR‐SU has antibacterial activity against S. mutans and also decreases S. mutans virulence.     

Phosphorylation and mRNA Splicing of Collapsin Response Mediator Protein-2 Determine Inhibition of Rho-associated Protein Kinase (ROCK) II Function in Carcinoma Cell Migration and Invasion

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.     

Identification of Mutations Involved in the Requirement of Potassium for Growth of Typical Melissococcus plutonius Strains

Melissococcus plutonius is a fastidious honeybee pathogen, and the addition of KH₂PO₄ to culture medium is required for its growth. Using genome sequences and a newly developed vector, we showed that mutations in genes encoding Na⁺/H⁺ antiporter and cation-transporting ATPase are involved in the potassium requirement for growth.     

Characterization and Modeling of Intermittent Locomotor Dynamics in Clock Gene-Deficient Mice

The scale-invariant and intermittent dynamics of animal behavior are attracting scientific interest. Recent findings concerning the statistical laws of behavioral organization shared between healthy humans and wild-type mice (WT) and their alterations in human depression patients and circadian clock gene (Period 2; Per2) mutant mice indicate that clock genes play functional roles in intermittent, ultradian locomotor dynamics. They also claim the clinical and biological importance of the laws as objective biobehavioral measures or endophenotypes for psychiatric disorders. In this study, to elucidate the roles of breakdown of the broader circadian regulatory circuit in intermittent behavioral dynamics, we studied the statistical properties and rhythmicity of locomotor activity in Per2 mutants and mice deficient in other clock genes (Bmal1, Clock). We performed wavelet analysis to examine circadian and ultradian rhythms and estimated the cumulative distributions of resting period durations during which locomotor activity levels are continuously lower than a predefined threshold value. The wavelet analysis revealed significant amplification of ultradian rhythms in the BMAL1-deficient mice, and instability in the Per2 mutants. The resting period distributions followed a power-law form in all mice. While the distributions for the BMAL1-deficient and Clock mutant mice were almost identical to those for the WT mice, with no significant differences in their parameter (power-law scaling exponent), only the Per2 mutant mice showed consistently and significantly lower values of the scaling exponent, indicating the increased intermittency in ultradian locomotor dynamics. Furthermore, based on a stochastic priority queuing model, we explained the power-law nature of resting period distributions, as well as its alterations shared with human depressive patients and Per2 mutant mice. Our findings lead to the development of a novel mathematical model for abnormal behaviors in psychiatric disorders.     

Asymmetrical Effect of Levodopa on the Neural Activity of Motor Regions in PD

Parkinson''s disease (PD) is a neurodegenerative illness often characterized by asymmetrical symptoms. However, the reason for this asymmetry and the cerebral correlates underlying symptom asymmetry are still not well understood. Furthermore, the effects of levodopa on the cerebral correlates of disease asymmetry have not been investigated. In this study, right-handed PD patients performed self-initiated, externally triggered and repetitive control finger movements with both their right and left hands during functional magnetic resonance imaging (fMRI) to investigate asymmetrical effects of levodopa on the hemodynamic correlates of finger movements. Patients completed two experimental sessions OFF and ON medication after a minimum of 12 hours medication withdrawal. We compared the effect of levodopa on the neural activation patterns underlying the execution of both the more affected and less affected hand for self-initiated and externally triggered movements. Our results show that levodopa led to larger differences in cerebral activity for movements of the more affected, left side: there were significant differences in activity after levodopa administration in regions of the motor cortico-striatal network when patients performed self-initiated and externally triggered movements with their left hand. By contrast, when patients used their right hand, levodopa led to differences in cerebellar activity only. As our patients were affected more severely on their left side, we propose that levodopa may help provide additional dopaminergic input, improving movements for the more severely affected side. These results suggest that the impact of reduced dopamine in the cortico-striatal system and the action of levodopa is not symmetrical.     

BAG-6 is essential for selective elimination of defective proteasomal substrates

BAG-6/Scythe/BAT3 is a ubiquitin-like protein that was originally reported to be the product of a novel gene located within the human major histocompatibility complex, although the mechanisms of its function remain largely obscure. Here, we demonstrate the involvement of BAG-6 in the degradation of a CL1 model defective protein substrate in mammalian cells. We show that BAG-6 is essential for not only model substrate degradation but also the ubiquitin-mediated metabolism of newly synthesized defective polypeptides. Furthermore, our in vivo and in vitro analysis shows that BAG-6 interacts physically with puromycin-labeled nascent chain polypeptides and regulates their proteasome-mediated degradation. Finally, we show that knockdown of BAG-6 results in the suppressed presentation of MHC class I on the cell surface, a procedure known to be affected by the efficiency of metabolism of defective ribosomal products. Therefore, we propose that BAG-6 is necessary for ubiquitin-mediated degradation of newly synthesized defective polypeptides.