The CD19 complex of B lymphocytes. Activation of phospholipase C by a protein tyrosine kinase-dependent pathway that can be enhanced by the membrane IgM complex.

We have investigated the mechanism by which the membrane protein complex of the B lymphocyte that contains CD19 and CR2 activates phospholipase C (PLC) to induce a rise in [CA2+]i. The CD19 complex resembled the membrane IgM complex in that three protein tyrosine kinase inhibitors suppressed increases in [Ca2+]i and inositol bisphosphate and inositol triphosphate generation. However, the activation of PLC by the CD19 complex could be distinguished from that by the membrane IgM complex by slower kinetics of generation of inositol phosphates, resistance to inhibition by activators of protein kinase C, and different pattern of tyrosine-phosphorylated cellular substrates. Western blot analysis of lysates from cells stimulated by the CD19 complex demonstrated a single new phosphotyrosine-containing protein of 85 kDa, whereas multiple other phosphotyrosine-containing proteins were present in cells activated by the mIgM complex. In particular, PLC-gamma 1, which is a substrate for the protein tyrosine kinase activated by the mIgM complex, was not tyrosine-phosphorylated in cells stimulated by the CD19 complex. Cross-linking the two complexes together caused a synergistic increase in [CA2+]i which was neither suppressed by activation of protein kinase C nor associated with increased tyrosine-phosphorylation of PLC, characteristic of the CD19 pathway. Therefore, the B cell has two signal transduction complexes, associated with membrane IgM and CD19, that activate PLC by different mechanisms and that can synergistically interact to enhance this function by the CD19 pathway.     

Structural and functional analysis of phytotoxin toxoflavin-degrading enzyme

Pathogenic bacteria synthesize and secrete toxic low molecular weight compounds as virulence factors. These microbial toxins play essential roles in the pathogenicity of bacteria in various hosts, and are emerging as targets for antivirulence strategies. Toxoflavin, a phytotoxin produced by Burkholderia glumae BGR1, has been known to be the key factor in rice grain rot and wilt in many field crops. Recently, toxoflavin-degrading enzyme (TxDE) was identified from Paenibacillus polymyxa JH2, thereby providing a possible antivirulence strategy for toxoflavin-mediated plant diseases. Here, we report the crystal structure of TxDE in the substrate-free form and in complex with toxoflavin, along with the results of a functional analysis. The overall structure of TxDE is similar to those of the vicinal oxygen chelate superfamily of metalloenzymes, despite the lack of apparent sequence identity. The active site is located at the end of the hydrophobic channel, 9 Å in length, and contains a Mn(II) ion interacting with one histidine residue, two glutamate residues, and three water molecules in an octahedral coordination. In the complex, toxoflavin binds in the hydrophobic active site, specifically the Mn(II)-coordination shell by replacing a ligating water molecule. A functional analysis indicated that TxDE catalyzes the degradation of toxoflavin in a manner dependent on oxygen, Mn(II), and the reducing agent dithiothreitol. These results provide the structural features of TxDE and the early events in catalysis.     

A Facile Synthesis of cis-9-[4-(1,2-Dihydroxyethyl)-cyclopent-2-enyl]guanine and Its Derivative

Abstract

The synthesis of carbocyclic nucleosides, cis-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine (3) and cis-2-amino-6-cyclopropylamino-9-[4-(1,2-dihydroxyethyl)-cyclopent-2-enyl]guanine (4), was achieved from cyclopentadiene (5) in five and six steps, respectively. This route involves a hetero Diels-Alder reaction and a Pd(0)-catalyzed coupling reaction.     

Targeted exon sequencing fails to identify rare coding variants with large effect in rheumatoid arthritis

Introduction

Although it has been suggested that rare coding variants could explain the substantial missing heritability, very few sequencing studies have been performed in rheumatoid arthritis (RA). We aimed to identify novel functional variants with rare to low frequency using targeted exon sequencing of RA in Korea.

Methods

We analyzed targeted exon sequencing data of 398 genes selected from a multifaceted approach in Korean RA patients (n = 1,217) and controls (n = 717). We conducted a single-marker association test and a gene-based analysis of rare variants. For meta-analysis or enrichment tests, we also used ethnically matched independent samples of Korean genome-wide association studies (GWAS) (n = 4,799) or immunochip data (n = 4,722).

Results

After stringent quality control, we analyzed 10,588 variants of 398 genes from 1,934 Korean RA case controls. We identified 13 nonsynonymous variants with nominal association in single-variant association tests. In a meta-analysis, we did not find any novel variant with genome-wide significance for RA risk. Using a gene-based approach, we identified 17 genes with nominal burden signals. Among them, VSTM1 showed the greatest association with RA (P = 7.80 × 10⁻⁴). In the enrichment test using Korean GWAS, although the significant signal appeared to be driven by total genic variants, we found no evidence for enriched association of coding variants only with RA.

Conclusions

We were unable to identify rare coding variants with large effect to explain the missing heritability for RA in the current targeted resequencing study. Our study raises skepticism about exon sequencing of targeted genes for complex diseases like RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0447-7) contains supplementary material, which is available to authorized users.     

Inhibitory effect of leptin on rosiglitazone-induced differentiation of primary adipocytes prepared from TallyHO/Jng mice

The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.