Regulatory Role for Complement Receptors (CD21/CD35) in the Recombination Activating Gene Expression in Mouse Peripheral B Cells

A population of peripheral B cells have been shown to express recombination activating gene products, RAG-1 and RAG-2, which are considered to be involved in revising the B cell antigen receptor (BCR) in the periphery. BCR engagement has been reported to turn off RAG expression in peripheral B cells, whereas the same treatment has an opposite effect in immature B cells in the bone marrow. In contrast to receptor editing that is involved in the removal of autoreactivity in immature B cells, it has been shown that secondary V(D)J rearrangement in peripheral B cells, termed receptor revision, contributes to affinity maturation of antibodies. Here, we show that RAG-2 expression in murine splenic B cells was abrogated by the coligation of BCR with complement receptors (CD21/CD35) much more efficiently than by the engagement of BCR alone. On the other hand, the same coligation augmented proliferation of anti-CD40-stimulated B cells. Consistent with these observations, RAG-2 expression was lower in the draining lymph nodes of the quasi-monoclonal mice when they were immunized with a high-affinity antigen than with a low-affinity one. These findings suggest a crucial role for CD21/CD35 in directing the conservation or the revision of BCRs in peripheral B cells

Generation of IgM and IgG1 monoclonal antibodies with identical variable regions: comparison of avidity

Generally, IgM antibodies (Abs) produced in a primary immune response show lower affinity for an inducing antigen (Ag) compared with the corresponding IgG Abs that are major switched isotypes formed in the secondary response. An IgM molecule is a pentamer with 10 Ag-binding sites that will contribute to an increase of avidity for an Ag. To estimate the contribution of the pentameric structure to the avidity of an IgM Ab, we generated IgM and IgG1 monoclonal Abs (mAbs) with identical V regions that are specific for 4-hydroxy-3-nitrophenylacetyl (NP) by in vitro class switching of B cells followed by the cell fusion with a mouse myeloma cell line. Compared with an anti-NP IgG1 mAb, the corresponding IgM mAb showed much higher avidity for NP-conjugated bovine serum albumin, which was drastically reduced after being dissociated into monomers

Cryptographic Pairings Based on Elliptic Nets

In 2007, Stange proposed a novel method of computing the Tate pairing on an elliptic curve over a finite field. This method is based on elliptic nets, which are maps from $\mathbb{Z}^n$ to a ring that satisfy a certain recurrence relation. In this paper, we explicitly give formulae for computing some variants of the Tate pairing: Ate, Ate$_i$, R-Ate and Optimal pairings, based on elliptic nets. We also discuss their efficiency by using some experimental results

Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line

During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2–3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling

Vision-based active sensor using a flexible beam

This paper proposes a new vision-based active sensing system, termed vision-based active antenna. This is composed of a camera, a flexible beam whose force-deformation characteristic is known, and an actuator for rotating the beam. The camera observes the beam deformation, including the contact information, while the beam is in contact with an object. By solving a set of equations based on the information acquired through the camera, the sensor can detect the contact location, the contact force, and the stiffness of the object, even though the contact point is hidden by occlusion. For two particular versions, we show some experimental results to verify the basic idea

Facile and rapid detection of toxic heavy metal ions using double-stranded DNA-gold nanoparticle conjugates containing mismatch base pair

課題番号：2170049

Optimised versions of the Ate and Twisted Ate Pairings

The Ate pairing and the twisted Ate pairing for ordinary elliptic curves which are generalizations of the $\eta_T$ pairing for supersingular curves have previously been proposed. It is not necessarily the case that both pairings are faster than the Tate pairing. In this paper we propose optimized versions of the Ate and twisted Ate pairings with the loop reduction method and show that both pairings are always at least as fast as the Tate pairing. We also provide suitable families of elliptic curves that our optimized Ate and optimized twisted Ate pairings can be computed with half the loop length compared to the Tate pairing

Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5′AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property

Regulation of Ca2+/calmodulin-dependent protein kinase kinase beta by cAMP signaling

BACKGROUND: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. METHODS: Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. RESULTS: Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. CONCLUSION: cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. GENERAL SIGNIFICANCE: Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation