Molecular characterization of two highly homologous receptor-like kinase genes, RLK902 and RKL1, in Arabidopsis thaliana

Receptor-like kinases (RLKs) constitute a large family of signal perception molecules. We characterized two highly homologous RLK genes, RLK902 and RKL1, in Arabidopsis. RLK902 and RKL1 showed a 75% amino acid sequence identity over their entire regions. In the RLK902 pro::GUS transgenic lines, GUS activity was strong in the root tips, lateral root primordia, stipules, and floral organ abscission zones, while the RKL1 promoter activity was dominant in the stomata cells, hydathodes and trichomes of young rosette leaves, and floral organ abscission zones. Neither the rlk902 mutant line, rkl1 mutant line nor rlk902/rkl1 double-knockout mutant line showed any significant phenotypes under normal growth conditions. These results suggest that RLK902 and RKL1 might mediate the signal transduction pathway in which at least one other complementary signaling pathway to these two RLKs might exist.     

G protein activation by endomorphins in the mouse periaqueductal gray matter

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.     

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34⁺CD38⁺CD33⁺ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3⁺ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.     

Exaggerated envy and guilt measured by economic games in Japanese women with anorexia nervosa

Background

Anorexia nervosa (AN) patients are assumed to express high levels of guilt and envy. Ultimatum game (UG) is a standard behavioral task that focuses on interpersonal behavior when splitting a sum of money between two players. UG studies consistently demonstrate that people tend to decrease their inequity in outcomes, one explanation being that economically irrational decision-making may partly arise from the emotions guilt and envy. We assumed that AN patients would perform excessively fair in UG, reflecting high guilt and envy.

Methods

We utilized UG to investigate the characteristics of guilt and envy among 24 Japanese AN patients and 22 age-matched healthy controls (HC). The relation between the outcome of UG and decision strategy confirmed by post-experimental questionnaires was analyzed.

Results

As proposer, AN offered a larger amount to the responder compared with HC (p?=?0.002) while, on the other hand, as responder, AN demanded much higher allocation to accept the offer compared with HC (p?=?0.026). Regarding the strategy as responder, AN put more emphasis on fairness and less emphasis on monetary reward compared with HC (p?=?0.046, p?=?0.042, respectively).

Conclusions

The results indicate that Japanese AN patients demonstrate strong preference for fairness, with high guilt and high envy. High sensitivity to guilt and envy of AN patients can affect not only their own behavior concerning eating attitude and body shape, but also decision-making in interpersonal situations. Behavioral experimental settings among social situations will enable us to evaluate and help actual decision-making in the real life of patients.

     

Sexual and temporal variations in floral scent in the subdioecious shrub Eurya japonica Thunb

In many flowering plants, floral scents are a significant trait for visitors, playing an important role in attracting pollinators and/or detracting herbivores. The evolution of flowering plants from hermaphroditism to dioecy is often accompanied by sexual dimorphism in floral scent. In this study, floral scents emitted by different sexual morphs of the subdioecious shrub Eurya japonica Thunb. were collected using a dynamic headspace method, and sexual and temporal variations were evaluated by gas chromatography–mass spectrometry (GC–MS). Two volatiles, α‐pinene and linalool, were identified as the major components of floral scents in females, hermaphrodites, and males. The males emit higher amounts of floral scents, particularly α‐pinene, compared to females or hermaphrodites. Floral scents emitted by males generally decrease as flowers enter senescence, whereas those from females or hermaphrodites do not significantly differ. Intraspecific variations in floral scents of subdioecious species provided by this study would contribute to better understanding of sexual dimorphism in floral scent.     

Comparison of cytoskeletal integrity,fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.     

Attenuation of fibroblast growth factor signaling by poly-N-acetyllactosamine type glycans

Fibroblast growth factors (FGFs) and their receptors are expressed in a variety of mammalian tissues, playing a role in development and cell proliferation. While analyzing human sperm motility, we found that sperm treated with endo-β-galactosidase (EBG), which specifically hydrolyzes poly-N-acetyllactosamine type glycans (polyLacs), enhanced motility. Mass spectrometry analysis revealed that sperm-associated polyLacs are heavily fucosylated, consistent with Lewis Y antigen. Immunohistochemistry of epididymis using an anti-Lewis Y antibody before and after EBG treatment suggested that polyLacs carrying the Lewis Y epitope are synthesized in epididymal epithelia and secreted to seminal fluid. EBG-treated sperm elevated cAMP levels and calcium influx, indicating activation of fibroblast growth factor signaling. Seminal fluid polyLacs bound to FGFs in vitro, and impaired FGF-mediated signaling in HEK293T cells.     

Rapid assay for detecting gyrA and parC mutations associated with fluoroquinolone resistance in Enterobacteriaceae

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.     

Dehydroepiandrosterone augments sensitivity to γ-ray irradiation in human H4 neuroglioma cells through down-regulation of Akt signaling

Dehydroepiandrosterone (DHEA) modulates sensitivity to radiation-induced injury in human neuroglioma cells (H4) through effects on Akt signalling by glutathione (GSH)-dependent redox regulation. Previous treatment of H4 cells with DHEA for 18 h reduced the γ-ray-induced phosphorylation of Akt, activated p21^waf1 synthesis and up-regulated phosphorylation of Rb independent of p53. These reactions were followed by a decrease in cell number and an increase in apoptosis and G₂/M checkpoint arrest. The suppression of phosphorylation of Akt by DHEA was due to regulation of the dephosphorylation by protein phosphatase 2A (PP2A). DHEA up-regulated the expression of γ-glutamylcysteine synthetase, a rate-limiting enzyme of glutathione (GSH) synthesis, and the levels of GSH to maintain PP2A activity. The results suggested that DHEA increases the sensitivity of cells to γ-ray irradiation by inducing apoptosis and cell cycle arrest through GSH-dependent regulation of the reduced form of PP2A to down-regulate the Akt signalling pathway.     

Characterization of Foam- and Emulsion-stabilizing Functions of Enzymatically Modified Proteins with Surfactancy

A papain-catalyzed reaction involving covalent incorporation of l-leucine n-alkyl ester is available for producing an enzymatically modified protein (EMP) with surfactancy [Agric. Biol. Chem., 45, 1621 (1981)]. In the present work we used gelatin as a starting material and incorporated l-leucine n-hexyl ester to produce a whippable EMP and l-leucine n-dodecyl ester to produce an emulsifiable EMP. A foam system formed with the whippable EMP was much stabler than that formed with sodium dodecylsulfate. The emulsifiable EMP also gave a much stabler oil-in-water emulsion than Tween-80 did. The stability of the emulsion formed with EMP was not affected by the presence of NaCl at a very high concentration. The observed foam and emulsion stabilities were well explained by the data for decreased mobility of the involved water protons. These results may indicate that EMP molecules, when arranged at the air/water or oil/water interface, can bind a part of the water to form thick barriers which prevent the air or oil particles from coalescing.