Specific recognition of linear polyubiquitin by A20 zinc finger 7 is involved in NF-κB regulation

LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-κB pathway through linear polyubiquitination of NEMO (NF-κB essential modulator, also known as IKKγ) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-κB activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-κB activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70–1.98 Å resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and IκB kinase (IKK), which results in NF-κB suppression. These findings provide new insight into the regulation of immune and inflammatory responses.     

Phytoplankton species abundance in Lake Kasumigaura (Japan) monitored monthly or biweekly since 1978

This data paper reports the abundance of phytoplankton species in monthly or biweekly samples collected from May 1978 through March 2010 at two stations on Lake Kasumigaura, a shallow lake that is the second-largest lake in Japan. The data set of quantitatively over several decades is unique among the available published data papers concerning lakes or plankton and continues to be freely available. The monitoring has been performed as a component of the Lake Kasumigaura Long-term Environmental Monitoring program, conducted by the National Institute for Environmental Studies since 1977. The data set details 173 phytoplankton species (or taxa), which can be identified by using an optical microscope and records their abundance. The abundance of each species is expressed in units of volume (??m³) per milliliter of lake water. This approach allows quantitative comparisons among taxa because the cell size of phytoplankton varies by several orders of magnitude among taxa. The phytoplankton data include 39 species (taxa) of Cyanophyta, 67 Chlorophyceae (Chlorophyta), 3 Prasinophyceae (Chlorophyta), 1 Raphidophyceae (Heterokontophyta), 6 Euglenophyceae (Euglenozoa), 4 Dinophyceae (Dinophyta), 38 Bacillariophyceae (Heterokontophyta), 6 Chrysophyceae (Heterokontophyta), 7 Xanthophyceae (Heterokontophyta), 1 Cryptophyceae (Cryptophyta) and 1 Prymnesiophyceae (Haptophyta). The data have been used for ecological and environmental studies and for studies on lake management.     

Loss of the maternal imprint in Dnmt3Lmat-/- mice leads to a differentiation defect in the extraembryonic tissue

DNA methylation of the genome is essential for mammalian development and plays crucial roles in a variety of biological processes including genomic imprinting. Although the DNA methyltransferase 3-like (Dnmt3L) protein lacks DNA methylase activity, it is thought to establish the maternal imprint in combination with the functional DNA methyltransferases. Oogenesis apparently proceeds normally in female mice homozygous for a targeted deletion of Dnmt3L, but their heterozygous offspring (Dnmt3L(mat-/-)) die before midgestation due to an imprinting defect. In this study, we show that Dnmt3L is required for the establishment of maternal methylation imprints both in the embryos and the placentae and that the placentae of these embryos develop abnormally. There is a defect in the formation of the labyrinth, reduced formation of the spongiotrophoblast layer, excess trophoblast giant cells and insufficient attachment between the chorion layer and the ectoplacental cone. In addition, we demonstrate arrest of proliferation of the extraembryonic tissue without apoptosis in vivo and a disturbance of the cell fate of Dnmt3L(mat-/-) trophoblastic stem cells in vitro. Furthermore, we report that DNA methylation during oogenesis is essential for the establishment of imprinting Mash2. These findings provide evidence that not only is DNA methylation required for the appropriate maternal imprint in the placenta but that the appropriate imprint is absolutely required for vertebrate placentation.     

Cutting edge: Uniqueness of lymphoid chemokine requirement for the initiation and maturation of nasopharynx-associated lymphoid tissue organogenesis

CD3(-)CD4(+)CD45(+) inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer's patches (PP) in the aerodigestive tract. CXCL13(-/-) mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3(-)CD4(+)CD45(+) cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13(-/-) and plt/plt mice, the formation of germinal centers in CXCL13(-/-) mice was affected, and their population of B cells was much lower than in the NALT of CXCL13(+/-) mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.     

Distribution of adherens junction mediated by VE-cadherin complex in rat spleen sinus endothelial cells

The splenic sinus endothelium regulates the passage of blood cells through the splenic cord. The goal of the present study was to assess the localization of vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin in the sinus endothelial cells of rat spleen and to characterize the presence and distribution of adherens junction formation mediated by the cadherin-catenin complex. Immunofluorescent microscopy of tissue cryosections demonstrated that VE-cadherin, β-catenin, and p120-catenin were localized in the junctional regions of adjacent endothelial cells. Double-staining immunofluorescent microscopy for VE-cadherin and β-catenin revealed colocalization at junctional regions. Transmission electron microscopy of thin sections of sinus endothelial cells treated with Triton X-100 clearly showed adherens junctions within the plasma membrane. Adherens junctions were located at various levels in the lateral membranes of adjacent endothelial cells regardless of the presence or absence of underlying ring fibers. Immunogold electron microscopy revealed VE-cadherin, β-catenin, and p120-catenin in the juxtaposed junctional membranes of adjacent sinus endothelial cells. Double-staining immunogold microscopy for VE-cadherin and β-catenin and for VE-cadherin and p120-catenin demonstrated colocalization to the junctional membranes of adjacent endothelial cells. Immunolabeling was evident at various levels in the lateral junctional membranes and was intermittently observed in the sinus endothelium. These data suggest that adherens junctions, whose formation appears to be mediated by VE-cadherin-catenin complexes, probably regulate the passage of blood cells through the spleen. This work was supported by a Grant-in-Aid for Scientific Research (C), Japan     

Disruption of phospholipase Cdelta4 gene modulates the liver regeneration in cooperation with nuclear protein kinase C

Phospholipase Cdelta4 (PLC delta4) gene has been cloned from the cDNA library of regenerating rat liver. Using PLC delta4 gene-disrupted mice (PLC delta4(-/-)), we studied a role of PLC delta4 during liver regeneration after partial hepatectomy (PH). In PLC delta4(-/-), liver regeneration occurred in an apparently normal way. However, BrdU-indices indicated that PLC delta4 gene disruption delayed the onset of DNA synthesis by 2 h. Noticeably, the BrdU-indices in PLC delta4(+/+) remained rather constant throughout S phase, 25-35%, whereas in PLC delta4(-/-), it fluctuated drastically from 25% at 34 h to 65% at late S, 42 h after PH. This fact showed that PLC delta4 gene disruption caused a higher synchronization of cell proliferation. The mRNA for PLC delta4 in PLC delta4(+/+) appeared at late G1, and the expression continued throughout S phase. PLC activity increased transiently in chromatin at the late G1 and S phases in only PLC delta4(+/+), but not in PLC delta4(-/-). The specific increases in PLC activity well correlated with the transient increases of protein kinase C (PKC) alpha in chromatin of PLC delta4(+/+). PKC epsilon also increased transiently in chromatin from PLC delta4(+/+) at late S. It is concluded that PLC delta4 regulates the liver regeneration in cooperation with nuclear PKC alpha and epsilon.     

The activity of a single muscle sympathetic vasoconstrictor nerve unit is affected by physiological stress in humans

Recording of neural firing from single-unit muscle sympathetic nerve activity (MSNA) is a new strategy offering information about the frequency of pure sympathetic firing. However, it is uncertain whether and when single-unit MSNA would be more useful than multiunit MSNA for analysis of various physiological stresses in humans. In 15 healthy subjects, we measured single-unit and multiunit MSNA before and during handgrip exercise at 30% of maximum voluntary contraction for 3 min and during the Valsalva maneuver at 40 mmHg expiratory pressure for 15 s. Shapes of individual single-unit MSNA were proved to be consistent and suitable for further evaluation. Single-unit and multiunit MSNA exhibited similar responses during handgrip exercise. However, acceleration of neural firing determined from single-unit MSNA became steeper than multiunit MSNA during the Valsalva maneuver. During the Valsalva maneuver, unlike handgrip exercise, the distribution of multiunit burst between 0, 1, 2, 3, and 4 spikes was significantly shifted toward multiple spikes within a given burst (P < 0.05). These results indicated that evaluation of single-unit MSNA could provide more detailed and accurate information concerning the role and responses of neuronal discharges induced by various physiological stresses in humans, especially amid intense sympathetic activity.     

Introduction and verification of a novel method for measuring wood fiber length using a single cross section in Acacia mangium

A novel method for calculating the wood fiber length using a single cross section was devised and verified in Acacia mangium. This method is based on the ratio of cell tips to total cell number in a cross section related to the wood fiber. The fiber length was calculated using the single cross-section method and was compared with the measurements obtained using the conventional maceration method and the serial cross-section method. There was no significant difference among the three methods.     

A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15–30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.     

Phosphorylation of tau at Ser214 mediates its interaction with 14-3-3 protein: implications for the mechanism of tau aggregation

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine –phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro . The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.