brachyenteron is necessary for morphogenesis of the posterior gut but not for anteroposterior axial elongation from the posterior growth zone in the intermediate-germband cricket Gryllus bimaculatus

In the long-germband insect Drosophila, all body segments and posterior terminal structures, including the posterior gut and anal pads, are specified at the blastoderm stage. In short- and intermediate-germband insects, however, posterior segments are sequentially produced from the posterior growth zone, a process resembling somitogenesis in vertebrates, and invagination of the posterior gut starts after anteroposterior (AP) axial elongation from the growth zone. The mechanisms underlying posterior segmentation and terminal patterning in these insects are poorly understood. In order to elucidate these mechanisms, we have investigated the roles of the Brachyury/brachyenteron (Bra/byn) homolog in the intermediate-germband cricket Gryllus bimaculatus. Loss-of-function analysis by RNA interference (RNAi) revealed that Gryllus byn (Gb'byn) is not required for AP axial elongation or normal segment formation, but is required for specification of the posterior gut. We also analyzed Gryllus caudal (Gb'cad) RNAi embryos using in situ hybridization with a Gb'byn probe, and found that Gb'cad is required for internalization of the posterior gut primordium, in addition to AP axial elongation. These results suggest that the functions of byn and cad in posterior terminal patterning are highly conserved in Gryllus and Drosophila despite their divergent posterior patterning. Moreover, because it is thought that the progressive growth of the AP axis from the growth zone, controlled by a genetic program involving Cdx/cad and Bra/byn, might be ancestral to bilaterians, our data suggest that the function of Bra/byn in this process might have been lost in insects.     

Function of the {alpha} Subunit of Rice Heterotrimeric G Protein in Brassinosteroid Signaling

The subunit of plant heterotrimeric G proteins (G) plays pivotalroles in multiple aspects of development and responses to planthormones. Recently, several lines of evidence have shown thatG participates in brassinosteroid (BR) responses in Arabidopsisand rice plants. In this study, we conducted a comprehensiveanalysis of the roles of the rice G in the responses to BR usinga defective mutant of the G gene, T65d1. Decreased sensitivityto 24-epi-brassinolide (24-epiBL) in the T65d1 mutant was observedin many processes examined, e.g. in the inhibition of root growthand the promotion of coleoptile elongation. The T65d1 mutantalso showed similar phenotypes to those of BR-deficient mutants,such as the specifically shortened second internode and theconstitutive photomorphogenic growth phenotype under dark conditions.However, a negative feedback effect by 24-epiBL on the expressionof BR biosynthetic genes was observed in the T65d1 mutant, andthe levels of BR intermediates did not fluctuate in this mutant.To determine the epistatic relationship between the T65d1 mutantand d61-7, a weak allele of a rice BR receptor mutant, the twomutants were crossed. The T65d1/d61-7 double mutant showed noepistasis in the elongation inhibition of the internodes, theinternode elongation pattern, the leaf angle and the morphologicalabnormality of leaf, except for the vertical length of seedand the seed weight. Our results suggest that the rice G affectsthe BR signaling cascade but the G may not be a signaling moleculein BRI1-meditated perception/transduction.     

Rational redesign of neutral endopeptidase binding to merlin and moesin proteins

Neutral endopeptidase (NEP) is a 90‐ to 110‐kDa cell‐surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti‐tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein–protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K¹⁹K²⁰K²¹ basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM‐related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E¹⁵⁹Q¹⁶⁰ in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N¹⁵⁹L¹⁶⁰ residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N¹⁵⁹L¹⁶⁰Y¹⁶¹K¹⁶²M¹⁶³ in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide–merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP‐binding protein. These data form the basis for further exploration of the details of NEP‐ERM binding and function.     

<Emphasis Type="Italic">S</Emphasis> locus-linked F-box genes expressed in anthers of <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S ₃ haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S ₃) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB511822–AB511825 and AB511859–AB511862.     

Synthesis of 11C-labeled 2-aminoethanol via a nitroaldol reaction using nitro[11C]methane

The nitroaldol reaction of nitro[¹¹C]methane and formaldehyde using EtOH and EtONa efficiently provided 2-nitro[¹¹C]ethanol in 3 min. The nitro group reduction in the presence of NiCl₂ and NaBH₄ in MeOH followed by purification using semi-preparative HPLC using 10% EtOH aqueous solution as an eluent proved to be a practical and accessible method for the synthesis of 2-amino[2-¹¹C]ethanol.     

Neutral endopeptidase is a myristoylated protein

Neutral endopeptidase (NEP) is a cell-surface peptidase normally expressed by prostate epithelial cells and lost in ~50% of primary prostate cancers. NEP directly associates with multiple proteins at the cell surface including Ezrin/Radixin/Moesin (ERM) proteins and the PTEN tumor suppressor protein. Analysis of the N-terminal sequence of the NEP cytosolic domain (N-terminal MGKSESQMDI TDINTPKPKK KQRWTR) identified a myristoylation consensus site. Mutation of Gly-2 to Arg significantly decreased ³H-myristoylation activity, and correlated with translocation of NEP from the plasma membrane to a perinuclear domain as demonstrated by immunofluorescence staining and Western blotting with an NEP-specific antibody. Removal of this myristoylation residue did not affect NEP enzymatic specific activity. Myristoylated NEP recruited more PTEN protein to the cell membrane fraction than unmyristoylated NEP. These data demonstrate that NEP is myristoylated at Gly-2 and that this modification is an intrinsic signal for membrane targeting.     

The Scaffold Protein Shoc2/SUR-8 Accelerates the Interaction of Ras and Raf

Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction.     

Engineered bio-nanocapsules, the selective vector for drug delivery system

The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.