Description: Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, β-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of β-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited β-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway.

Description: When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.

Description: In tropical dry forests, uppermost-canopy leaves of evergreen trees possess the ability to use water more conservatively compared with drought-deciduous trees, which may result from significant differences in the photoprotective mechanisms between functional types. We examined the seasonal variations in leaf gas exchange, chlorophyll fluorescence and the amounts of photosynthetic pigments within lamina of the uppermost-canopy leaves of three drought-deciduous trees ( Vitex peduncularis Wall., Xylia xylocarpa (Roxb.) W. Theob., Shorea siamensis Miq.), a semi-deciduous tree ( Irvingia malayana Miq.) and two evergreen trees ( Hopea ferrea Lanessan and Syzygium cumini (L.) Skeels) in Thailand. Area-based maximum carbon assimilation rates ( A max ) decreased during the dry season, except in S. siamensis . The electron transport rate (ETR) remained unchanged in deciduous trees, but decreased during the dry season in evergreen and semi-deciduous trees. In the principal component analysis, the first axis (Axis 1) accounted for 44.3% of the total variation and distinguished deciduous from evergreen trees. Along Axis 1, evergreen trees were characterized by a high Stern–Volmer non-photochemical quenching coefficient (NPQ), high xanthophyll cycle pigments/chlorophyll and a high de-epoxidation state of the xanthophyll cycle, whereas the deciduous trees were characterized by a high ETR, a high quantum yield of PSII ( PSII = ( F m ' – F )/ F m ' ) and a high mass-based A max under high-light conditions. These findings indicate that drought-deciduous trees showing less conservative water use tend to dissipate a large proportion of electron flow through photosynthesis or alternative pathways. In contrast, the evergreens showed more conservative water use, reduced A max and ETR and enhanced NPQ and xanthophyll cycle pigments/chlorophyll during the dry season, indicating that down-regulated photosynthesis with enhanced thermal dissipation of excess light energy played an important role in photoprotection. Trees with different water uses and leaf lifespans appear to employ different photoprotective mechanisms to overcome the unfavorable dry-season drought. Our data may suggest that future changes in precipitation will strongly impinge on forest structure and functions.

Description: Mucin 1 (MUC1) is overexpressed in various human malignant tumors and its expression is correlated with a poor prognosis. MUC1 engages in signal transduction by interacting with receptors for growth and differentiation factors, which contributes to the growth and survival of cancer cells. However, the mechanism by which MUC1 promotes cancer cell invasion remains unclear. Microarray analysis revealed that expression of urokinase-type plasminogen activator (uPA) was elevated in MUC1-overexpressing cells. Furthermore, up- and down-modulation of MUC1 expression was clearly correlated with the change of uPA expression. An immunochemical study showed that the distribution of uPA coincided with that of MUC1 in various human cancer tissues. The MUC1 C-terminal domain (MUC1-CD) was associated with nuclear factor-κB (NF-κB) p65 in MUC1-expressing cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that MUC1-CD existed with NF-κB p65 on the uPA promoter. Luciferase assays indicated that the uPA transcriptional activity was correlated with the level of MUC1 expression and that this MUC1-enhancing effect on the uPA transcription was abolished by introduction of mutations into the NF-κB binding sites on the uPA promoter. These results indicate that formation of the MUC1-CD and NF-κB p65 complex enhanced nuclear translocation of NF-κB p65 and subsequent occupancy of NF-κB binding region on the uPA promoter, leading to elevated transcription of uPA. We also demonstrated that uPA induced by MUC1 enhanced the matrix metalloproteinase (MMP)-2 and -9 activities, and consequently promoted cancer cell invasion. Thus, a MUC1 co-operating NF-κB signaling pathway plays a critical role in cancer cell invasion in MUC1-expressing cells.

Description: Multifunctional Ca 2+ /calmodulin-dependent protein kinases (CaMKs) have been extensively studied in mammals, whereas fungus CaMKs still remain largely uncharacterized. We previously obtained CaMK homolog in Coprinopsis cinerea , designated CoPK12, and revealed its unique catalytic properties in comparison with the mammalian CaMKs. To further clarify the regulatory mechanisms of CoPK12, we investigated post-translational modification and subcellular localization of CoPK12 in this study. In C. cinerea , full-length CoPK12 (65 kDa) was fractionated in the membrane fraction, while the catalytically active fragment (46 kDa) of CoPK12 was solely detected in the soluble fraction by differential centrifugation. Expressed CoPK12-GFP was localized on the cytoplasmic and vacuolar membranes as visualized by green fluorescence in yeast cells. In vitro N-myristoylation assay revealed that CoPK12 is N-myristoylated at Gly-2 in the N-terminal position. Furthermore, calmodulin could bind not only to CaM-binding domain but also to the N-terminal myristoyl moiety of CoPK12. These results, taken together, suggest that the cellular localization and function of CoPK12 are regulated by protein N-myristoylation and limited proteolysis.

Description: The frequency of extreme weather has been rising in recent years. A 3-year study of street trees was undertaken in Tokyo to determine whether: (i) street trees suffer from severe water stress in unusually hot summer; (ii) species respond differently to such climatic fluctuations; and (iii) street trees are also affected by nitrogen (N) deficiency, photoinhibition and aerosol pollution. During the study period (2010–12), midsummers of 2010 and 2012 were unusually hot (2.4–2.8 °C higher maximum temperature than the long-term mean) and dry (6–56% precipitation of the mean). In all species, street trees exhibited substantially decreased photosynthetic rate in the extremely hot summer in 2012 compared with the average summer in 2011. However, because of a more conservative stomatal regulation (stomatal closure at higher leaf water potential) in the hot summer, apparent symptoms of hydraulic failure were not observed in street trees even in 2012. Compared with Prunus  x  yedoensis and Zelkova serrata , Ginkgo biloba , a gymnosperm, was high in stomatal conductance and midday leaf water potential even under street conditions in the unusually hot summer, suggesting that the species had higher drought resistance than the other species and was less susceptible to urban street conditions. This lower susceptibility might be ascribed to the combination of higher soil-to-leaf hydraulic conductance and more conservative water use. Aside from meteorological conditions, N deficiency affected street trees significantly, whereas photoinhibition and aerosol pollution had little effect. The internal CO 2 and 13 C suggested that both water and N limited the net photosynthetic rate of street trees simultaneously, but water was more limiting. From these results, we concluded that the potential risk of hydraulic failure caused by climatic extremes could be low in urban street trees in temperate regions. However, the size of the safety margin might be different between species.