Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis

Mammary glands, like other skin appendages such as hair follicles and teeth, develop from the surface epithelium and underlying mesenchyme; however, the molecular controls of embryonic mammary development are largely unknown. We find that activation of the canonical WNT/beta-catenin signaling pathway in the embryonic mouse mammary region coincides with initiation of mammary morphogenesis, and that WNT pathway activity subsequently localizes to mammary placodes and buds. Several Wnt genes are broadly expressed in the surface epithelium at the time of mammary initiation, and expression of additional Wnt and WNT pathway genes localizes to the mammary lines and placodes as they develop. Embryos cultured in medium containing WNT3A or the WNT pathway activator lithium chloride (LiCl) display accelerated formation of expanded placodes, and LiCl induces the formation of ectopic placode-like structures that show elevated expression of the placode marker Wnt10b. Conversely, expression of the secreted WNT inhibitor Dickkopf 1 in transgenic embryo surface epithelium in vivo completely blocks mammary placode formation and prevents localized expression of all mammary placode markers tested. These data indicate that WNT signaling promotes placode development and is required for initiation of mammary gland morphogenesis. WNT signals play similar roles in hair follicle formation and thus may be broadly required for induction of skin appendage morphogenesis.     

IL-4 differentially regulates eotaxin and MCP-4 in lung epithelium and circulating mononuclear cells

To investigate the mechanisms of eosinophil recruitment in allergic airway inflammation, we examined the effects of interleukin (IL)-4, a Th2-type cytokine, on eotaxin and monocyte chemoattractant protein-4 (MCP-4) expression in human peripheral blood mononuclear cells (PBMCs; n = 10), in human lower airway mononuclear cells (n = 5), in the human lung epithelial cell lines A549 and BEAS-2B, and in human cultured airway epithelial cells. IL-4 inhibited eotaxin and MCP-4 mRNA expression induced by IL-1 beta and tumor necrosis factor-alpha in PBMCs but did not significantly inhibit expression in epithelial cells. Eotaxin and MCP-4 mRNA expression was not significantly induced by proinflammatory cytokines in lower airway mononuclear cells. IL-1 beta-induced eotaxin and MCP-4 protein production was also inhibited by IL-4 in PBMCs, whereas IL-4 enhanced eotaxin protein production in A549 cells. In contrast, dexamethasone inhibited eotaxin and MCP-4 expression in both PBMCs and epithelial cells. The divergent effects of IL-4 on eotaxin and MCP-4 expression between PBMCs and epithelial cells may create chemokine concentration gradients between the subepithelial layer and the capillary spaces that may promote the recruitment of eosinophils to the airway in Th2-type responses.     

DOES ELASTIN CONTRIBUTE TO THE PERSISTENCE OF CORPORA ALBICANTIA IN THE OVARY OF THE COMMON DOLPHIN (DELPHINUS DELPHIS)

The corpora albicantia (CAs) from the ovaries of 39 common dolphins ( Delphinus delphis ) caught in driving fisheries were used, and histologically and immunohistochemically examined. The area of each corpus albicans (CA) and the proportion of that area occupied by elastin were measured using NIH-image software. In all CAs, elastoid material (EM) was apparent although EM area varied in each CA. CAs increased in number with dolphin age. Smaller CAs contained a higher proportion of EM. EM was completely digested by elastase, but not by collagenase. Furthermore, EM was immunostained with anti-α-elastin antibody. These results demonstrated that EM was elastin. The present study is the first to describe the presence of elastin in cetacean CAs. The higher proportion of elastin in small-sized CAs of common dolphins is suggested as the likely cause of the persistence of CA.     

Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint

Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast. We have found that the spindle checkpoint kinase Bub1p becomes phosphorylated by Cdc2p during spindle damage in mitotic cells. Cdc2p directly phosphorylates Bub1p in vitro at the CDK consensus sites. A Bub1p mutant that cannot be phosphorylated by Cdc2p is checkpoint defective, indicating that Cdc2p-dependent Bub1p phosphorylation is required to activate the checkpoint after spindle damage. The kinase activity of Bub1p is required, but is not sufficient, for complete spindle checkpoint function. The role of Bub1p in maintaining centromeric localization of Rec8p during meiosis I is entirely dependent upon its kinase activity, suggesting that Bub1p kinase activity is essential for establishing proper kinetochore function. Finally, we show that there is a Bub1p-dependent meiotic checkpoint, which is activated in recombination mutants.     

Erythrina poeppigiana-derived phytochemical exhibiting antimicrobial activity against Candida albicans and methicillin-resistant Staphylococcus aureus

AIMS: To screen five phytochemicals isolated from Erythrina poeppigiana (Leguminosae) for antimicrobial activity against both Candida albicans and methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: Roots of E. poeppigiana were macerated with acetone and the chloroform-soluble fraction of the residue was subjected to repeated silica gel column chromatography using various eluting solvents. Structures of the isolated compounds were determined by extensive spectroscopic studies. Each compound was dissolved in dimethyl sulphoxide and added to agar plates (final concentration: 1.56-100 microg ml(-1)) and minimum inhibitory concentrations (MICs) against C. albicans and MRSA were determined. Spectral data indicated the presence of three different types of phytochemicals; isoflavonoids (erypoegin A, demethylmedicarpin and sandwicensin), alpha-methyldeoxybenzoin (angolensin) and cinnamylphenol (erypostyrene). While all compounds showed anti-MRSA activity in this concentration range, isoflavonoids and alpha-methyldeoxybenzoin failed to inhibit the growth of C. albicans. Erypostyrene (E-1-[2-hydroxy-4-methoxy-5-(gamma,gamma-dimethylallyl)benzyl]-2-(4-hydroxyphenyl)ethylene) exhibited not only the highest anti-MRSA activity (MIC value of 6.25 microg ml(-1)) but also anti-candidal potency (MIC value of 50 microg ml(-1)). The compound reduced viable cell numbers of C. albicans and MRSA by approximately 1 of 2000 and 1 of 1000 after 1 h incubation at each MIC, respectively. CONCLUSIONS: A new cinnamylphenol, erypostyrene, possessed anti-candidal and anti-MRSA activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Erypostyrene could be a leading candidate for development of antimicrobial agents with anti-candidal and anti-MRSA activity.     

Measuring adsorption of a hydrophobic probe with a surface plasmon resonance sensor to monitor conformational changes in immobilized proteins

Conformational changes of proteins immobilized on solid matrices were observed by measuring the adsorption of Triton X-100 (TX), a nonionic detergent, as a hydrophobic probe with BIACORE, a biosensor that utilizes the phenomenon of surface plasmon resonance (SPR). Two kinds of proteins, alpha-glucosidase and lysozyme, were covalently attached to dextran matrices on the sensor surface in the flow cell and then exposed to various concentrations of TX solution. We measured SPR signal changes derived from adsorption of TX to the immobilized proteins and calculated the monolayer adsorption capacity using the Brunauer-Emmett-Teller (BET) equation. The results demonstrated that monolayer adsorption capacity is proportional to the amount of immobilized proteins. Further, the unfolding process of immobilized proteins on the sensor surface induced by guanidine hydrochloride was investigated by monitoring SPR signal increases due to the adsorption of TX to the exposed hydrophobic region of the protein. Results strongly suggested that the increase in the SPR signal reflected the formation of the agglutinative unfolded state. We expect our measuring method using the SPR sensor and TX adsorption will be a novel tool to provide conformational information regarding various proteins on solid matrices.     

Specific expression of annexin III in rat-small-hepatocytes

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.     

Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants

Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.