Identification of amino acid substitutions that render the Arabidopsis cytokinin receptor histidine kinase AHK4 constitutively active

In Arabidopsis, three genes (AHK2, AHK3 and AHK4/CRE1) encode histidine kinases (His-kinases), which serve as cytokinin receptors. To understand how the external cytokinin signal activates the His-kinase across the cell membrane, we exploited the power of microbial genetics to isolate several AHK4 mutants that function independently of cytokinin in both prokaryotic and eukaryotic assay systems. In each mutant, a single amino acid substitution within the second membrane-spanning segment, or within the region around the phosphorylation His site, renders the His-kinase constitutively active. These mutant receptors appear to have a 'locked-on' conformation, even in the absence of stimulus. We discuss the implications of these data for the structure and function of the cytokinin receptor His-kinases in plants.     

Establishment of monokaryotic and dikaryotic isolates of Hedgehog mushrooms (Hydnum repandum and related species) from basidiospores

Hydnum repandum and its relatives are gourmet edible ectomycorrhizal mushrooms. However, no reliable pure cultures have been reported in this genus. Here, we report for the first time the successful isolation of mycelial strains from basidiospores in the genus Hydnum. Basidiospores obtained from basidioma samples were aseptically inoculated onto modified Norkran's C (MNC) medium, MNC containing n-butyric acid (n-MNC), or MNC with gellan gum instead of agar (G-MNC). Although basidiospore germination was observed in most samples, the isolation rate was higher from MNC (91.7%) and G-MNC (93.8%) than from n-MNC (36.4%). Most established isolates were monokaryotic and lacked a clamp connection, but three were dikaryotic and had clamp connections. Established isolates were identified by molecular phylogenetic analysis of the internal transcribed spacer region of the rRNA gene. These results suggest that basidiospores can be used to establish monokaryotic and dikaryotic isolates of Hydnum species.     

Motilin Stimulates Gastric Acid Secretion in Coordination with Ghrelin in Suncus murinus

Motilin and ghrelin constitute a peptide family, and these hormones are important for the regulation of gastrointestinal motility. In this study, we examined the effect of motilin and ghrelin on gastric acid secretion in anesthetized suncus (house musk shrew, Suncus murinus), a ghrelin- and motilin-producing mammal. We first established a gastric lumen-perfusion system in the suncus and confirmed that intravenous (i.v.) administration of histamine (1 mg/kg body weight) stimulated acid secretion. Motilin (0.1, 1.0, and 10 μg/kg BW) stimulated the acid output in a dose-dependent manner in suncus, whereas ghrelin (0.1, 1.0, and 10 μg/kg BW) alone did not induce acid output. Furthermore, in comparison with the vehicle administration, the co-administration of low-dose (1 μg/kg BW) motilin and ghrelin significantly stimulated gastric acid secretion, whereas either motilin (1 μg/kg BW) or ghrelin (1 μg/kg BW) alone did not significantly induce gastric acid secretion. This indicates an additive role of ghrelin in motilin-induced gastric acid secretion. We then investigated the pathways of motilin/motilin and ghrelin-stimulated acid secretion using receptor antagonists. Treatment with YM 022 (a CCK-B receptor antagonist) and atropine (a muscarinic acetylcholine receptor antagonist) had no effect on motilin or motilin-ghrelin co-administration-induced acid output. In contrast, famotidine (a histamine H₂ receptor antagonist) completely inhibited motilin-stimulated acid secretion and co-administration of motilin and ghrelin induced gastric acid output. This is the first report demonstrating that motilin stimulates gastric secretion in mammals. Our results also suggest that motilin and co-administration of motilin and ghrelin stimulate gastric acid secretion via the histamine-mediated pathway in suncus.     

Substrate selectivity of epidermal growth factor-receptor ligand sheddases and their regulation by phorbol esters and calcium influx

Signaling via the epidermal growth factor receptor (EGFR), which has critical roles in development and diseases such as cancer, is regulated by proteolytic shedding of its membrane-tethered ligands. Sheddases for EGFR-ligands are therefore key signaling switches in the EGFR pathway. Here, we determined which ADAMs (a disintegrin and metalloprotease) can shed various EGFR-ligands, and we analyzed the regulation of EGFR-ligand shedding by two commonly used stimuli, phorbol esters and calcium influx. Phorbol esters predominantly activate ADAM17, thereby triggering a burst of shedding of EGFR-ligands from a late secretory pathway compartment. Calcium influx stimulates ADAM10, requiring its cytoplasmic domain. However, calcium influx-stimulated shedding of transforming growth factor alpha and amphiregulin does not require ADAM17, even though ADAM17 is essential for phorbol ester-stimulated shedding of these EGFR-ligands. This study provides new insight into the machinery responsible for EGFR-ligand release and thus EGFR signaling and demonstrates that dysregulated EGFR-ligand shedding may be caused by increased expression of constitutively active sheddases or activation of different sheddases by distinct stimuli.     

Immunization with dendritic cells loaded with alpha-galactosylceramide at priming phase, but not at boosting phase, enhances cytotoxic T lymphocyte activity against infection by intracellular bacteria

We evaluated the effect of immunization with dendritic cells (DCs) pulsed with alpha-galactosylceramide (alphaGalCer) and listeriolysin O (LLO) 91-99 peptide, a dominant cytotoxic T lymphocyte (CTL) epitope of Listeria monocytogenes by observing the responses of specific CD8(+) T cells and in vivo CTL activity. DCs were pulsed with various combinations of alphaGalCer and LLO91-99 peptide and administered to BALB/c mice. Immunization with DCs pulsed with alphaGalCer and LLO91-99 at priming phase and with DCs pulsed with LLO91-99 alone at boosting phase induced stronger in vivo CTL activity, reduced the bacterial load in spleens of Listeria-challenged mice and augmented CD62L(+) CD8(+) central memory T cells compared with other immunization protocols. The blockade of interferon-gamma (IFN-gamma) at boosting phase reversed the induction of CD8(+) central memory T cells and reduced the bacterial load in spleens of Listeria-challenged mice immunized with DCs pulsed with alphaGalCer and LLO91-99 at both phases, suggesting that alphaGalCer at boosting phase has deleterious effects through IFN-gamma production. These results indicate that immunization with DCs pulsed with CTL epitope peptide together with alphaGalCer at priming phase, but not at boosting phase, is feasible for eliciting a specific CTL activity and protective immunity against infection of intracellular bacteria.     

Requirement for ERK MAP kinase in mouse preimplantation development

Preimplantation development is a crucial step for successful implantation and pregnancy. Although both compaction and blastocyst formation have been extensively studied, mechanisms regulating the early cell division stages before compaction have remained unclear. Here, we show that extracellular signal regulated kinase (ERK) mitogen-activated protein (MAP) kinase function is required for early embryonic cell division before compaction. Our analysis demonstrates that inhibition of ERK activation in late two-cell-stage embryos leads to a reversible arrest in the G2 phase at the four-cell stage. The G2-arrested four-cell-stage embryos showed weakened cell-cell adhesion as compared with control embryos. Remarkably, microarray analyses showed that most of the programmed changes of upregulated and downregulated gene expression during the four- to eight-cell stages proceeded normally in four-cell-stage-arrested embryos that were subsequently released to resume development; however, the expression profiles of a proportion of genes in these embryos closely paralleled the stages of embryonic rather than normal development. These parallel genes included the genes encoding intercellular adhesion molecules, whose expression appeared to be positively regulated by the ERK pathway. We also show that, whereas ERK inactivation in eight-cell-stage embryos did not lead to cell division arrest, it did cause this arrest when cadherin-mediated cell-cell adhesion was disrupted. These results demonstrate an essential role of ERK function in two-cell to eight-cell-stage embryos, and suggest a loose parallelism between the gene expression programs and the developmental stages before compaction.     

The Peniophorella praetermissa species complex (Basidiomycota)

The corticioid basidiomycete Peniophorella praetermissa has long been regarded as a morphologically variable species complex. An ITS-based phylogenetic study based on a worldwide sampling was carried out using parsimony and Bayesian inference. The resulting trees feature three major clades, further divided into well-supported subclades. These could be considered as distinct species, a contention that is further supported by crossing test data. Only two out of the eight phylogenetic lineages identified can be distinctly morphologically characterized: P. odontiaeformis and P. subpraetermissa. P. odontiaeformis is an odontioid species with a paleotropical distribution whereas the taxa in the remaining subclades have smooth basidiomata and are distributed in temperate areas. P. subpraetermissa is known only from the type collection and is distinguished microscopically by its reddish brown apically encrustated cystidia. Taxa in the remaining subclades are impossible to distinguish from each other morphologically, and therefore, are viewed as a species complex, P. praetermissa s. lat. One of the subclades, which is widely distributed but restricted to the Northern hemisphere, is proposed to represent P. praetermissa s. str. An epitype is selected from the same area as the holotype, among the specimens studied here. However, the geographically most widespread clade with many representatives from both hemispheres is here referred to as P. pertenuis, a taxon that was previously considered a synonym to P. praetermissa.