Embryonic Medaka Model of Microglia in the Developing CNS Allowing In Vivo Analysis of Their Spatiotemporal Recruitment in Response to Irradiation

Radiation therapy (RT) is pivotal in the treatment of many central nervous system (CNS) pathologies; however, exposure to RT in children is associated with a higher risk of secondary CNS tumors. Although recent research interest has focused on the reparative and therapeutic role of microglia, their recruitment following RT has not been elucidated, especially in the developing CNS. Here, we investigated the spatiotemporal dynamics of microglia during tissue repair in the irradiated embryonic medaka brain by whole-mount in situ hybridization using a probe for Apolipoprotein E (ApoE), a marker for activated microglia in teleosts. Three-dimensional imaging of the distribution of ApoE-expressing microglia in the irradiated embryonic brain clearly showed that ApoE-expressing microglia were abundant only in the late phase of phagocytosis during tissue repair induced by irradiation, while few microglia expressed ApoE in the initial phase of phagocytosis. This strongly suggests that ApoE has a significant function in the late phase of phagocytosis by microglia in the medaka brain. In addition, the distribution of microglia in p53-deficient embryos at the late phase of phagocytosis was almost the same as in wild-type embryos, despite the low numbers of irradiation-induced apoptotic neurons, suggesting that constant numbers of activated microglia were recruited at the late phase of phagocytosis irrespective of the extent of neuronal injury. This medaka model of microglia demonstrated specific recruitment after irradiation in the developing CNS and could provide a useful potential therapeutic strategy to counteract the detrimental effects of RT.     

Biogas production using anaerobic groundwater containing a subterranean microbial community associated with the accretionary prism

In a deep aquifer associated with an accretionary prism, significant methane (CH₄) is produced by a subterranean microbial community. Here, we developed bioreactors for producing CH₄ and hydrogen (H₂) using anaerobic groundwater collected from the deep aquifer. To generate CH₄, the anaerobic groundwater amended with organic substrates was incubated in the bioreactor. At first, H₂ was detected and accumulated in the gas phase of the bioreactor. After the H₂ decreased, rapid CH₄ production was observed. Phylogenetic analysis targeting 16S rRNA genes revealed that the H₂-producing fermentative bacterium and hydrogenotrophic methanogen were predominant in the reactor. The results suggested that syntrophic biodegradation of organic substrates by the H₂-producing fermentative bacterium and the hydrogenotrophic methanogen contributed to the CH₄ production. For H₂ production, the anaerobic groundwater, amended with organic substrates and an inhibitor of methanogens (2-bromoethanesulfonate), was incubated in a bioreactor. After incubation for 24 h, H₂ was detected from the gas phase of the bioreactor and accumulated. Bacterial 16S rRNA gene analysis suggested the dominance of the H₂-producing fermentative bacterium in the reactor. Our study demonstrated a simple and rapid CH₄ and H₂ production utilizing anaerobic groundwater containing an active subterranean microbial community.     

Clinical Features and Treatment Modes of Mandibular Fracture at the Department of Oral and Maxillofacial Surgery,Shimane University Hospital,Japan

Background

The number of elderly patients with maxillofacial trauma is rapidly increasing due to active lifestyles and longevity. Shimane prefecture has the fastest growing proportion of elderly individuals in Japan. The aim of this study was to reveal the distinctive features and treatment modes of mandibular fracture treatment mode in patients requiring hospitalization at the Department of Oral and Maxillofacial Surgery, Shimane University Hospital, Japan.

Patients and Methods

Patient age, sex, period between injury and first consultation, years since injury, cause of injury, fracture site, treatment, and duration of hospitalization were evaluated. Univariate Poisson regression, relative risk with 95% confidence interval based on the Wald test, Fisher’s exact test, and Kruskal-Wallis test were used to explore associations among clinical and demographic variables.

Results

In total, 305 patients were diagnosed with and hospitalized for mandibular fracture from 1980 to 2010. Younger age increased the risk for mandibular fracture. Incidence was higher in males than females, particularly in the young, but the male to female ratio decreased with age. The period until first hospital consultation decreased progressively over the study period. Fall was a much more frequent cause in patients aged ≥60 than in those aged <60 years. Mandibular fracture with condyle, symphysis, and angle involvement were most common and were associated with sex, age, and treatment mode. Length of hospitalization has decreased since 1980.

Conclusion

In our department, patients aged ≥60 years accounted for a greater proportion of mandibular fracture cases than in many previous studies, reflecting the greater proportion of elderly residents in Shimane prefecture.     

Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway

The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2–3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.     

Activation of cytosolic Slingshot-1 phosphatase by gelsolin-generated soluble actin filaments

Slingshot-1 (SSH1) is a protein phosphatase that dephosphorylates and activates cofilin, an actin-severing and -disassembling protein. SSH1 is bound to and activated by F-actin, but not G-actin. SSH1 is accumulated in the F-actin-rich lamellipodium but is also diffusely distributed in the cytoplasm. It remains unknown whether SSH1 is activated by soluble (low-level polymerized) actin filaments in the cytoplasm. In this study, we show that SSH1 binds to gelsolin via actin filaments in the cytosolic fraction. Gelsolin promoted solubilization of actin filaments and SSH1 in cell-free assays and in cultured cells. SSH1 was activated by gelsolin-generated soluble actin filaments. Furthermore, gelsolin enhanced cofilin dephosphorylation in neuregulin-stimulated cells. Our results suggest that cytosolic SSH1 forms a complex with gelsolin via soluble actin filaments and is activated by gelsolin-generated soluble actin filaments and that gelsolin promotes stimulus-induced cofilin dephosphorylation through increasing soluble actin filaments, which support SSH1 activation in the cytoplasm.     

A Highlights from MBoC Selection: Involvement of p114-RhoGEF and Lfc in Wnt-3a– and Dishevelled-Induced RhoA Activation and Neurite Retraction in N1E-115 Mouse Neuroblastoma Cells

The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a–induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF–silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a–induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a–induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a– and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.     

Phylogenomic Analyses Reveal the Evolutionary Origin of the Inhibin α-Subunit,a Unique TGFβ Superfamily Antagonist

Transforming growth factor-beta (TGFβ) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFβ superfamily that share a common β-subunit. The evolution of the inhibin α-subunit created the only antagonist within the TGFβ superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin α-subunit appear to reflect specific insertion-deletion changes within the inhibin β-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFβ superfamily.     

Interannual variations in abundance and body size in Neocalanus copepods in the central North Pacific

As the integral components of zooplankton in the subarctic NorthPacific, the three Neocalanus species (N. cristatus, N. plumchrusand N. flemingeri) are characterized by an annual life cycleand rapid development in the surface layer during spring–summer.Patterns of interannual variation of abundance and body sizeof these Neocalanus species were analyzed using the time-seriesdata collected during the summers of 1979–1998 (20 years)at stations along the longitudinal transect line in the centralNorth Pacific, crossing five sub-areas (Alaska Current System,Subarctic Current System, Northern Transition Domain, SouthernTransition Domain and Subtropical Current System). In the southernsub-areas, quasi-decadal oscillation was observed for the 3-yearrunning mean of abundance and prosome length for copepoditestage 5 (C5) of the three Neocalanus species. Although the oscillationsignal diminished towards northern waters, it showed a positivephase during the early 1980s and 1990s and a negative phaseduring the late 1980s. In the northern waters, a biennial patternwas pronounced for anomalies of C5 prosome length for N. plumchrusand N. flemingeri, which was large in odd years and small ineven years. Significantly positive covariations among the threespecies were found for both abundance and prosome length aroundmid-latitude, where they were abundant. In the correlation analysis,these observed yearly patterns showed a statistically insignificantcorrelation with most environmental (integrated mean temperaturein surface waters, water column stability and chlorophyll aconcentration) or climatological (North Pacific Index and SouthernOscillation Index) variables. The regional difference of theoscillation signal and the synchronized covariation among thesespecies suggest that interannual variations of their abundanceand body size are mediated by common environmental force(s)with some spatial and temporal scales in the subarctic NorthPacific.     

DNA adduct formation in human hepatoma cells treated with 3-nitrobenzanthrone: analysis by the (32)P-postlabeling method

3-Nitrobenzanthrone (3-nitro-7H-benz[d,e]anthracen-7-one, 3-NBA) is a powerful mutagen and a suspected human carcinogen existing in diesel exhaust and airborne particulates. Recently, one of the major presumed metabolites of 3-NBA, 3-aminobenzanthrone (3-ABA), was detected in human urine samples. Here we analyzed DNA adducts formed in 3-NBA-exposed human hepatoma HepG2 cells by a (32)P-postlabeling/thin layer chromatography (TLC) method and a (32)P-postlabeling/polyacrylamide gel electrophoresis (PAGE) method. With HepG2 cells exposed to 3-NBA (0.36-36.4 microM) for 3h, we obtained three spots or bands corresponding to adducted nucleotides. Two were assigned as 2-(2'-deoxyadenosin-N(6)-yl)-3-aminobenzanthrone-3'-phosphate (dA3'p-N(6)-C2-ABA) and 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone-3'-phosphate (dG3'p-N(2)-C2-ABA), with identical mobilities to those of synthetic standards on PAGE analysis. The chemical structure of the substance corresponding to the other spot or band could not be identified. Quantitative analyses revealed that the major adduct was dA3'p-N(6)-C2-ABA and its relative adduct labeling (RAL) value at 36.4 microM of 3-NBA was 200.8+/-86.1/10(8)nucleotide.     

Myosin Vb is required for trafficking of the cystic fibrosis transmembrane conductance regulator in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.