Defective immune responses in mice lacking LUBAC‐mediated linear ubiquitination in B cells

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF‐κB pathway, which is important for B‐cell development and function. Here, we describe a mouse model (B‐HOIP^Δlinear) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF‐κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B‐HOIP^Δlinear mice due to defective activation of the IKK complex; however, B‐cell receptor (BCR)‐mediated activation of the NF‐κB and ERK pathways was unaffected. B‐HOIP^Δlinear mice show impaired B1‐cell development and defective antibody responses to thymus‐dependent and thymus‐independent II antigens. Taken together, these data suggest that LUBAC‐mediated linear polyubiquitination is essential for B‐cell development and activation, possibly via canonical NF‐κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.     

Effect of morph types, body size and prior residence on food-site holding by males of the male-dimorphic stag beetle Prosopocoilus inclinatus (Coleoptera: Lucanidae)

Mate-securing tactics of small males in male-polymorphic species exhibiting male–male combat is an important issue in behavioral ecology. While most studies have focused on the outcomes of such combat encounters, the holding of a mating resource like a feeding site has a greater impact for obtaining reproductive success. We examined the effects of the prior residence at a feeding site on resource acquisition in the male-dimorphic stag beetle, Prosopocoilus inclinatus. More than 70 % of encounters did not result in combat. While larger males tended to occupy a food site after a combat, smaller males with prior residence tended to occupy food sites when no fighting occurred. Morph types or body size have no effect on the occurrence of combat, meaning that small males do not hesitate to fight with large males. These findings show that, under experimental conditions, the prior residence has a positive effect to hold food site in P. inclinatus.     

Dynamical Properties of Condensed Charged Polymer Melts

Abstract

Dynamical properties of condensed charged polymer melts are studied with a two-dimensional model and molecular dynamics simulation. Screened Coulombic interactions are assumed for the interactions between the monomer charges as well as the counterions which were introduced to neutralize the total monomer charges of polymer chains. Through molecular dynamics calculations, we have obtained the radial distribution function and velocity auto-correlation function, and their density dependences. As a structural characteristics in condensed charged polymer melts, we find that the monomers tend to form triangular structures locally. The radial distribution function for the center monomers implies that the polymer chains on our two-dimensional model are not entangled. The diffusion properties for both the counter-ions and innermost monomers are studied in detail. In this paper, we have also computed neutral polymer melts to study the effects of the presence of the long-range Coulombic interactions on the dynamical properties of polymer melts. We find that the Coulombic interactions significantly reduce the self diffusion. Snapshots analyses obtained from the molecular dynamics simulation suggest that the reptation model is not a proper model for two-dimensional polymer-chain melts, in which there are little entanglement effects in agreement with the result of the analyses for the radial distribution function.     

Seasonal Dynamics of Dimethylarsenic Acid Degrading Bacteria Dominated in Lake Kibagata

Degradation processes of organoarsenic compounds significantly influence arsenic cycles in aquatic environments and would depend on the bacterial activities. The bacterial population involving dimethylarsinic acid (DMAA) degradation was investigated in Lake Kibagata from April to December in 2003. During the experimental period, the methylated arsenic was not detected, although the inorganic arsenic concentration ranged from 3.4 nM to 9.2 nM. Moreover, in the sample water of Lake Kibagata to which DMAA added, DMAA decreased while inorganic arsenic increased for 25 days. These facts suggested that the bacteria remineralized methylate arsenic species to inorganic arsenic. In fact, monitoring the use of Most Probable Number (MPN) procedure demonstrated that the DMAA-degrading bacteria exist at cell densities ranged from 41 cells/ml to 510 cells/ml. To determine the composition of DMAA-degrading bacteria, the total 110 isolates obtained as dominated bacterial species were analyzed by the restriction-fragment-length polymorphism (RFLP) analysis of 16S rDNA. As a result, total 110 isolates were classified into 12 types, of which 4 types dominated during the spring and/or fall seasons, and the rest 8 types dominated during summer season. DMAA degrading activities of the 110 isolates ranged at various degrees. Especially, the some isolates of fall season tend to show high degradation activities. The phylogenetic analysis using 16S rDNA revealed that the representative isolates formed several clusters in the gram-positive bacterial group and the proteobacteria subdivision. The diverse compositions of DMAA-degrading bacteria would seasonally change to control the rates of organoarsenic degradation in Kibagata.     

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti‐ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C‐terminal src kinase)‐binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent‐resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti‐ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over‐expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314‐phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314‐phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP‐43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.     

Cortical GluK1 kainate receptors modulate scratching in adult mice

Recent investigations into the mechanisms mediating itch transmission have focused on spinal mechanisms, whereas few studies have investigated the role of the cerebral cortex in itch‐related behaviors. Human imaging studies show that several cortical regions are active in correspondence with itch, including the anterior cingulate cortex (ACC). We present here evidence of cortical modulation of pruritogen‐induced scratching behavior. We combine pharmacological, genetic, and electrophysiological approaches to show that cortical GluK1‐containing kainate (KA) receptors are involved in scratching induced by histamine and non‐histamine‐dependent itching stimuli. We further show that scratching corresponds with enhanced excitatory transmission in the ACC through KA receptor modulation of inhibitory circuitry. In addition, we found that inhibiting GluK1‐containing KA receptors in the ACC also reduced behavioral nociceptive responses induced by formalin. Our results reveal a new role of the cortex in pruritogen‐induced scratching.

     

Isomerase Pin1 Stimulates Dephosphorylation of Tau Protein at Cyclin-dependent Kinase (Cdk5)-dependent Alzheimer Phosphorylation Sites

Neurodegenerative diseases associated with the pathological aggregation of microtubule-associated protein Tau are classified as tauopathies. Alzheimer disease, the most common tauopathy, is characterized by neurofibrillary tangles that are mainly composed of abnormally phosphorylated Tau. Similar hyperphosphorylated Tau lesions are found in patients with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) that is induced by mutations within the tau gene. To further understand the etiology of tauopathies, it will be important to elucidate the mechanism underlying Tau hyperphosphorylation. Tau phosphorylation occurs mainly at proline-directed Ser/Thr sites, which are targeted by protein kinases such as GSK3β and Cdk5. We reported previously that dephosphorylation of Tau at Cdk5-mediated sites was enhanced by Pin1, a peptidyl-prolyl isomerase that stimulates dephosphorylation at proline-directed sites by protein phosphatase 2A. Pin1 deficiency is suggested to cause Tau hyperphosphorylation in Alzheimer disease. Up to the present, Pin1 binding was only shown for two Tau phosphorylation sites (Thr-212 and Thr-231) despite the presence of many more hyperphosphorylated sites. Here, we analyzed the interaction of Pin1 with Tau phosphorylated by Cdk5-p25 using a GST pulldown assay and Biacore approach. We found that Pin1 binds and stimulates dephosphorylation of Tau at all Cdk5-mediated sites (Ser-202, Thr-205, Ser-235, and Ser-404). Furthermore, FTDP-17 mutant Tau (P301L or R406W) showed slightly weaker Pin1 binding than non-mutated Tau, suggesting that FTDP-17 mutations induce hyperphosphorylation by reducing the interaction between Pin1 and Tau. Together, these results indicate that Pin1 is generally involved in the regulation of Tau hyperphosphorylation and hence the etiology of tauopathies.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.