Structural element responsible for the Fe(III)-phytosiderophore specific transport by HvYS1 transporter in barley

Hordeum vulgare L. yellow stripe 1 (HvYS1) is a selective transporter for Fe(III)-phytosiderophores, involved in primary iron acquisition from soils in barley roots. In contrast, Zea mays yellow stripe 1 (ZmYS1) in maize possesses broad substrate specificity, despite a high homology with HvYS1. Here we revealed, by assessing the transport activity of a series of HvYS1-ZmYS1 chimeras, that the outer membrane loop between the sixth and seventh transmembrane regions is essential for substrate specificity. Circular dichroism spectra indicated that a synthetic peptide corresponding to the loop of HvYS1 forms an alpha-helix in solution, whereas that of ZmYS1 is flexible. We propose that the structural difference at this particular loop determines the substrate specificity of the HvYS1 transporter.     

Rod1, an arrestin-related protein, is phosphorylated by Snf1-kinase in Saccharomyces cerevisiae

Metazoan arrestin proteins bind to seven-transmembrane proteins, mediate their internalization and play central roles in the subsequent signal transduction pathway. In Saccharomyces cerevisiae, there are several arrestin-related proteins. One of those proteins, Rod1, has been identified to have the ability to confer resistance to o-dinitrobenzene. We found that Rod1 interacted with Snf4, a subunit of Snf1-kinase complex. Both snf4 and snf1 mutants were also sensitive to the drug and the kinase activity of Snf1 was required for the drug tolerance. In immunoblotting analysis, the Rod1 protein was phosphorylated in an Snf1-dependent manner in vivo, and the phosphorylation of the serine residue 447 of Rod1 was responsible for the band-shift. Furthermore, the Rod1 protein was directly phosphorylated by Snf1-kinase in vitro. The substitution of the serine residue 447 to alanine slightly enhanced the resistance to the drug. We discuss possible functions of Rod1.     

Essential role of the N-terminus of murine Orai1 in store-operated Ca2+ entry

Store-operated Ca(2+) entry (SOCE) is a physiologically important process that is triggered by intracellular Ca(2+) depletion. Recently, human Orai1 (the channel-forming subunit) and STIM1 (the calcium sensor) were identified as essential molecules for SOCE. Here, we report the cloning and functional analysis of three murine orthologs of Orai1, termed Orai1, 2, and 3. Among the genes identified, Orai1 contains a distinctive proline- and arginine-rich N-terminal cytoplasmic sequence. Co-expression of STIM1 with Orai1 produced a marked effect on SOCE, while co-expression with Orai2 or Orai3 had little effect. Expression of Orai1 without its N-terminal tail had a marginal effect on SOCE, while chimeric Orai2 containing the Orai1 N-terminus produced a marked increase in SOCE. In addition, a truncated version of Orai1 containing the N-terminus without the pore-forming transmembrane domain had a dominant negative effect on SOCE. These results reveal the essential role of Orai1 and its N-terminal sequence in SOCE.     

<Emphasis Type="Italic">π</Emphasis>–<Emphasis Type="Italic">π</Emphasis> interaction between aromatic ring and copper-coordinated His81 imidazole regulates the blue copper active-site structure

Noncovalent weak interactions play important roles in biological systems. In particular, such interactions in the second coordination shell of metal ions in proteins may modulate the structure and reactivity of the metal ion site in functionally significant ways. Recently, π–π interactions between metal ion coordinated histidine imidazoles and aromatic amino acids have been recognized as potentially important contributors to the properties of metal ion sites. In this paper we demonstrate that in pseudoazurin (a blue copper protein) the π–π interaction between a coordinated histidine imidazole ring and the side chains of aromatic amino acids in the second coordination sphere, significantly influences the properties of the blue copper site. Electronic absorption and electron paramagnetic resonance spectra indicate that the blue copper electronic structure is perturbed, as is the redox potential, by the introduction of a second coordination shell π–π interaction. We suggest that the π–π interaction with the metal ion coordinated histidine imidazole ring modulates the electron delocalization in the active site, and that such interactions may be functionally important in refining the reactivity of blue copper sites. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A wave of EGFR signaling determines cell alignment and intercalation in the Drosophila tracheal placode

Invagination of organ placodes converts flat epithelia into three-dimensional organs. Cell tracing in the Drosophila tracheal placode revealed that, in the 30-minute period before invagination, cells enter mitotic quiescence and form short rows that encircle the future invagination site. The cells in the rows align to form a smooth boundary (;boundary smoothing'), accompanied by a transient increase in myosin at the boundary and cell intercalation oriented in parallel with the cellular rows. Cells then undergo apical constriction and invaginate, followed by radially oriented mitosis in the placode. Prior to invagination, ERK MAP kinase is activated in an outward circular wave, with the wave front often correlating with the smoothing cell boundaries. EGFR signaling is required for myosin accumulation and cell boundary smoothing, suggesting its propagation polarizes the planar cell rearrangement in the tracheal placode, and coordinates the timing and position of intrinsic cell internalization activities.     

Dendritic cell‐targeting DNA‐based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag‐specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant‐based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA‐based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag‐specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine‐keyhole limpet hemocyanin (PC‐KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae . Finally, the possibility that anti‐PC antibodies induced by nasal delivery of pFL plus PC‐KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.

     

Potential for interspecific hybridization between <Emphasis Type="Italic">Zizina emelina</Emphasis> and <Emphasis Type="Italic">Zizina otis</Emphasis> (Lepidoptera: Lycaenidae)

Environmental changes such as global warming and biological invasion caused by human activities raise the possibility of secondary contact between the endangered butterfly species Zizina emelina and its sibling species Zizina otis in Japan. To assess the possible risks from their habitats overlapping, we investigated the potential for hybridization and the development of F1 individuals. We observed successful mating of the two sibling species under artificial conditions. The presence of a postzygotic hybridization barrier was supported by the delay of larval development only in females; a delay did not occur in males. Existence of the barrier was also supported by a decreased egg hatching rate in one brood; this was likely associated with infection with Wolbachia, a bacterium manipulating the reproductive capability of its host. The size and wing markings of F1 hybrid individuals were intermediate between those of the two species. These results suggest that, if Z. emelina and Z. otis are distributed sympatrically in the future, there is a possibility of introgression and reproductive interference between the two species, which would increase the risk of decline of each species.     

Newly raised anti‐VAChT and anti‐ChAT antibodies detect cholinergic cells in chicken embryos

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O‐acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.