Insecticidal activities of methyleugenol and β-asarone,from the herbal medicines Saishin and Sekishōkon,and other alkoxy-propenyl-benzene derivatives against the cigarette beetle <Emphasis Type="Italic">Lasioderma serricorne</Emphasis> (Coleoptera: Anobiidae)

The principal insecticidal compounds from the herbal medicines Saishin, the root of Asarum sieboldii Miquel, and Sekishōkon, the rhizome of Acorus gramineus Soland, were isolated by successive silica gel column chromatography and high performance liquid chromatography. The active components, which work against larvae of the cigarette beetle, Lasioderma serricorne (F.), were identified as methyleugenol (4-allyl-1,2-dimethoxybenzene) and β-asarone {1,2,4-trimethoxy-5-[(Z)-prop-1-enyl]benzene} by gas chromatography and gas chromatography/mass spectrometry analyses. These two compounds share some structural features, such as a benzene ring with o-dimethoxy groups and a propenyl group in the opposite position. Consequently, the insecticidal activities of 20 structurally related compounds were tested to evaluate their structure–activity relationship. We found myristicin (5-allyl-1-methoxy-2,3-methylenedioxybenzene) exhibited comparable insecticidal activity to methyleugenol and β-asarone, but the other tested compounds were less active. The lack of insecticidal activity of compounds with a hydrogen, hydroxy, or acetoxy substituent in place of one methoxy group indicates that o-dimethoxy groups are essential for insecticidal activity. The position and configuration of a double bond in the propenyl side chain affected the toxicity, but there was a lack of consistency in the structure–activity relationship for this.     

Release of 6-keto-prostaglandin F1α and thromboxane B2 from mouse peritoneal macrophages during their adhesion and spreading on a glass surface

The release of 6-keto-prostaglandin F_1α (6KF_1α)_and of thromboxane B₂ (TXB₂) from cells were investigated using mouse peritoneal exudate cells (PECs) and non-cultured peritoneal macrophages. They were prepared by adhesion to glass dishes and incubated for 1 hr at 37°C in 5% Co₂ in air. Both the percentage of spreading macrophages and the release of 6KF_1α and TXb₂ increased in proportion to the incubation time. 6KF_1α and TXB₂ were released from the macrophages, not from the non-adherent cells. When PECs were incubated in silicon-coated glass dishes, the spreading of macrophages was hardly detected and lower amounts of 6KF_1α and TXB₂ were released from these cells compared with cells incubated in non-treated glass dishes. These findings suggest that adhesion with the correlated spreading of macrophages on glass dishes serve as a considerable physical factor for the release of 6KF_1α and TXB₂.     

Onset of amnesia is delayed with a decrease in inhibition of protein synthesis during odor-taste associative learning in the terrestrial slug Limax valentianus

Slugs can retain odor-taste associative memory for several weeks, and this requires protein synthesis. We examined the dose-dependency of the onset time of amnesia caused by the protein synthesis inhibitor anisomycin, and showed that with reduced dose, the onset is shifted from 2 days to 3 days after conditioning; we could not shift the onset delay later than 3 days. Our results suggest that the mechanism underling memory retention is different in the period up to 3 days, versus the period later than 3 days. Our results also suggest that sustained inhibition of protein synthesis in the period from zero to 3 hr after conditioning is necessary to cause amnesia.     

Stiffness of γ subunit of F1-ATPase

F₁-ATPase is a molecular motor in which the γ subunit rotates inside the α₃β₃ ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F₁-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F₁ and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F₁ from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α₃β₃ ring, and the complex of the external part of the γ subunit and the α₃β₃ ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F_o and F₁-ATPase.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

Defective relocalization of ALS2/alsin missense mutants to Rac1-induced macropinosomes accounts for loss of their cellular function and leads to disturbed amphisome formation

Loss of ALS2/alsin function accounts for several recessive motor neuron diseases. ALS2 is a Rab5 activator and its endosomal localization is regulated by Rac1 via macropinocytosis. Here, we show that the pathogenic missense ALS2 mutants fail to be localized to Rac1-induced macropinosomes as well as endosomes, which leads to loss of the ALS2 function as a Rab5 activator on endosomes. Further, these mutants lose the competence to enhance the formation of amphisomes, the hybrid-organelle formed upon fusion between autophagosomes and endosomes. Thus, Rac1-induced relocalization of ALS2 might be crucial to exert the ALS2 function associated with the autophagy-endolysosomal degradative pathway.     

Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation

Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides.