Mating behavior and the function of the male genital spine in the ground beetle Carabus clathratus

The morphologies of male genitalia often appear harmful or aggressive, as if they may inflict physical damage upon females during copulation. Such male genitalia are often thought to function in intra- and intersexual interactions during mating. In the carabid genus Carabus, division Spinulati, males possess a spine (spinula) on the intromittent organ, of which function is unknown. To reveal the function of the spinula, we studied the mating behavior and genital coupling of a Spinulati species, Carabus (Limnocarabus) clathratus. The males positioned the spinula along the inner wall of the vaginal opening throughout copulation. This placement created a small dent and subsequently a melanized patch (wound) on the vaginal wall, but the spinula rarely penetrated the vaginal wall. The spinula did not reach the innermost part of the vagina where the spermatophore is deposited. These results suggest that the spinula is not used for inflicting damage on female genitalia or manipulating spermatophores of rival males. During spermatophore formation, the male partially withdrew the aedeagus, and only the aedeagal tip and endophallus remained within the female. By placing the spinula against the vaginal wall, the male could hold the endophallus within the vaginal chamber in the unstable copulatory posture. Thus, our observations suggest that the spinula primarily functions as an "anchor" to maintain the coupling of the male and female genitalia and thereby ensure insemination.     

The smallest active fragment of microtubule-associated protein 4 and its interaction with microtubules in phosphate buffer

To analyze the interaction between microtubule-associated protein (MAP) 4 and microtubules physicochemically, a MAP4 active site fragment was designed for nuclear magnetic resonance (NMR) use. The fragment was bacterially expressed and purified to homogeneity. The buffer conditions for NMR were optimized to support microtubule assembly. The fragment was found to bind to microtubules under the optimized buffer conditions.     

Centaurin-alpha1 is a phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-activated protein kinases

Centaurin-alpha1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-alpha1 is still not understood. Here we have shown that transient expression of centaurin-alpha1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-alpha1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-alpha1-dependent ERK activation. Furthermore, transient knockdown of centaurin-alpha1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-alpha1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3.     

Methylation of Smad6 by protein arginine N-methyltransferase 1

Signal transduction pathways utilize posttranslational modifications to regulate the activity of their components in a temporal-spatial and efficient fashion. Arginine methylation is one of the posttranslational modifications that can result in monomethylated-, asymmetric dimethylated- and/or symmetric dimethylated-arginine residues in proteins. Here we demonstrate that inhibitory-Smads (Smad6 and Smad7), but not receptor-regulated- (R-)Smads and the common-partner Smad4, can be methylated by protein arginine N-methyltransferase (PRMT)1. Using mass-spectrometric analysis, we found that PRMT1 dimethylates arginine(74) (Arg(74)) in mouse Smad6. PRMT1 interacts with the N-terminal domain of Smad6 in which Arg(74) residue is located. Assays examined so far have shown no significant differences between the functions of Smad6 and those of methylation-defective Smad6 (Smad6R74A). Both wild-type and Smad6R74A were equally efficient in blocking BMP-induced growth arrest upon their ectopic expression in HS-72 mouse B-cell hybridoma cells.     

Characterization and tissue distribution of multiple agouti-family genes in pufferfish, Takifugu rubripes

Four types of agouti-family genes (AGRP1, AGRP2, ASIP1 and ASIP2) were obtained from torafugu, Takifugu rubripes. Their characterization and structure were analyzed to elucidate the relationship among the torafugu agouti-family genes. Both AGRP1 and AGRP2 showed genomic synteny with the human AGRP gene. Phylogenetic tree analysis showed that AGRP1 formed a cluster with human AGRP. We inferred that torafugu AGRP1 and AGRP2 are orthologs of human AGRP and that they are paralogous genes derived from genome duplication occurred in the teleost phylogeny. Torafugu ASIP1 showed genomic synteny with the human ASIP, but ASIP2 did not. The ASIP1 expression level was about five times higher in the white ventral skin than in the black dorsal skin. Therefore, we concluded that torafugu ASIP1 is an ortholog of human ASIP, nevertheless, we are unable to determine if torafugu ASIP2 is a paralog of ASIP1 or not.     

Characterization of multimetric variants of ubiquitin carboxyl-terminal hydrolase L1 in water by small-angle neutron scattering

Here, we illustrated that the morphological structures of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) variants and Parkinson's disease (PD) exhibit good pathological correlation by a small-angle neutron scattering (SANS). UCH-L1 is a neuro-specific multiple functional enzyme, deubiquitinating, ubiquityl ligase, and also involved in stabilization of mono-ubiquitin. To examine the relationship between multiple functions of UCH-L1 and the configuration of its variants [wild-type, I93M (linked to familial Parkinson's disease), and S18Y (linked to reduced risk of Parkinson's disease)], in this report, we proposed that these were all self-assembled dimers by an application of a rotating ellipsoidal model; the configurations of these dimers were quite different. The wild-type was a rotating ellipsoidal. The globular form of the monomeric component deformed by the I93M mutation. Conversely, the S18Y polymorphism promoted the globularity. Thus, the multiple functional balance is closely linked to the intermolecular interactions between the UCH-L1 monomer and the final dimeric configuration.