Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1. Mice lacking PLA2G3, L-PGDS or DP1, mast cell-deficient mice reconstituted with PLA2G3-null or DP1-null mast cells, or mast cells cultured with L-PGDS-ablated fibroblasts exhibited impaired maturation and anaphylaxis of mast cells. Thus, we describe a lipid-driven PLA2G3-L-PGDS-DP1 loop that drives mast cell maturation.

Hypervalent Iodine(III)-Mediated Decarboxylative Ritter-Type Amination Leading to the Production of α-Tertiary Amine Derivatives.

α-Tertiary amines (ATAs) are attractive structural motifs that are frequently found in biologically active molecules. Therefore, the development of an efficient method for the synthesis of ATAs represents an important research topic in the field of medicinal chemistry as well as organic chemistry. Although the Ritter reaction is a reliable approach for preparing α-tertiary amine derivatives via intermolecular amination reactions, the typical methods suffer from disadvantages such as harsh reaction conditions and the use of strong acids. Because of this, it has been of limited use in the synthesis of ATAs. We report here on the decarboxylative Ritter-type amination of carboxylic acids bearing an α-quaternary carbon center using a combination of PhI(OAc)2 and molecular iodine (I2) to produce the corresponding α-tertiary amine derivatives. This reaction proceeded at ambient temperature on the benchtop with a fluorescent light. Mechanistic investigations suggest that the reaction proceeds via the formation of an alkyl iodide and a higher oxidation state iodine(III) species as key intermediates. Similarly, a stepwise protocol for the Ritter-type amination of alcohols via the formation of oxalic acid monoalkyl esters was also achieved. The present methods represent a useful tool for the synthesis of ATAs that are difficult to prepare by conventional methods.

Ubiquitous versus restricted expression of the two mouse dendritic cell C-type lectin receptors, DCIR1 and DCAR2, among myeloid cells.

Dendritic cell inhibitory receptor 1 (DCIR1, also known as DCIR and Clec4a2) and dendritic cell activating receptor 2 (DCAR2, also known as DCAR and Clec4b1) are mouse lectin receptors expressed on antigen presenting cells. They have structurally similar C-type lectin domains, of which amino acid sequences show 90.5% identity, and commercially available antibodies against them cross-react each other. Here we have established novel antibodies against DCIR1 and DCAR2 that can unambiguously discriminate DCIR1 and DCAR2 and examined their distribution among various immune cells. While DCIR1 was ubiquitously expressed on myeloid cells, including conventional DCs (cDCs), macrophages, neutrophils and eosinophils, in various immune organs, significant expression of DCAR2 was detected only on subpopulations of cDCs from bone marrow and skin-draining lymph nodes. Interestingly, in FITC-painted mice, DCAR2 was expressed on all of the FITC(+) cDCs, which had migrated from the skin after FITC painting, suggesting that DCAR2 can be a marker of migratory cDCs in skin-draining lymph nodes. Our findings provide a basis to investigate in vivo function of DCIR1 and DCAR2.

Iodine-Catalyzed Decarboxylative Amidation of ß,γ-Unsaturated Carboxylic Acids with Chloramine Salts Leading to Allylic Amides.

The iodine-catalyzed decarboxylative amidation of ß,γ-unsaturated carboxylic acids with chloramine salts is described. This method enables the regioselective synthesis of allylic amides from various types of ß,γ-unsaturated carboxylic acids containing substituents at the α- and ß-positions. In the reaction, N-iodo-N-chloroamides, generated by the reaction of a chloramine salt with I2 , function as a key active species. The reaction provides an attractive alternative to existing methods for the synthesis of useful secondary allylic amine derivatives.

Synthesis and Structure of Hypervalent Iodine(III) Reagents Containing Phthalimidate and Application to Oxidative Amination Reactions.

A new class of hypervalent iodine reagents containing phthalimidate was synthesized, and structurally characterized by X-ray analysis. The benziodoxole-based reagent displays satisfactory solubility in common organic solvents and is reasonably stable in solution as well as in the solid state. The reagent was used for the oxidative amination of the C(sp(3))-H bond of N,N-dimethylanilines. In addition, the reagent was also applicable to oxidative amination with rearrangement of trialkylamines as well as enamines that were prepared in situ from secondary amines and aldehydes.

Production of anti-carbohydrate antibodies by phage display technologies: potential impairment of cell growth as a result of endogenous expression.

Anti-mannotriose (Man3) antibodies were previously isolated from a Keio phage library displaying human single chain variable fragments (scFvs) using a neoglycolipid, Man3- dipalmitoylphosphatidylethanolamine. Of three genes constructed, the 5A3 clone was expressed in mouse myeloma NS0 cells as a conjugate with human IgG(1) Fc (scFv-Fc) and characterized (Sakai, K., Shimizu, Y., Chiba, T., Matsumoto-Takasaki, A., Kusada, Y., Zhang, W., Nakata, M., Kojima, N., Toma, K., Takayanagi, A., Shimizu, N., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 253-262; Zhang, W., Matsumoto-Takasaki, A., Kusada, Y., Sakaue, H., Sakai, K., Nakata, M., and Fujita-Yamaguchi, Y. (2007) Biochemistry 46, 263-270). Similarly, anti-Le(x) phages were screened from the same library with lacto-N-fucopentaose III (LNFPIII; Le(x))-dipalmitoylphosphatidylethanolamine. Of five phage clones isolated, two scFv genes were constructed to express scFv-Fc proteins in NS0 cells. As was experienced with anti-Man3 scFv-Fc clones, only one anti-LNFPIII clone, 1F12, was successfully produced and purified as an scFv-Fc protein. Although anti-LNFPIII 1F12 and anti-Man3 5A3 scFv-Fc proteins were secreted into media, a decline in scFv-Fc production was observed with both stable clones during early passages. Transient expression of anti-LNFPIII and anti-Man3 scFv-Fc genes in COS-7 cells and subsequent analyses of scFv-Fc protein expression revealed accumulation of translated proteins in the endoplasmic reticulum for scFv-Fc proteins derived from clones that did not survive as stable clones. This report describes the following: (i) isolation of anti-LNFPIII scFv genes; (ii) purification of anti-LNFPIII scFv-Fc proteins from stably and transiently expressed cells; and (iii) extracellular or intracellular localization of two anti-LNFPIII and three anti-Man3 scFv-Fc proteins. The results suggest that expression of anti-Man3 and other anti-carbohydrate antibodies in mammalian cells is disadvantageous for cell growth.

Transcriptomic signature of the follicular somatic compartment surrounding an oocyte with high developmental competence.

During antral folliculogenesis, developmental competence of prospective oocytes is regulated in large part by the follicular somatic component to prepare the oocyte for the final stage of maturation and subsequent embryo development. The underlying molecular mechanisms are poorly understood. Oocytes reaching the advanced stage of follicular growth by administration of exogenous follicle-stimulating hormone (FSH) possess higher developmental competence than oocytes in FSH-untreated smaller follicles. In this study, the transcriptomic profile of the cumulus cells from cows receiving FSH administration (FSH-priming) was compared, as a model of high oocyte competence, with that from untreated donor cows (control). Ingenuity Pathway Analysis showed that cumulus cells receiving FSH-priming were rich in down-regulated transcripts associated with cell movement and migration, including the extracellular matrix-related transcripts, probably preventing the disruption of cell-to-cell contacts. Interestingly, the transcriptomic profile of up-regulated genes in the control group was similar to that of granulosa cells from atretic follicles. Interferon regulatory factor 7 was activated as the key upstream regulator of FSH-priming. Thus, acquisition of developmental competence by oocytes can be ensured by the integrity of cumulus cells involved in cell-to-cell communication and cell survival, which may help achieve enhanced oocyte-somatic cell coupling.

Crystal structure of human dendritic cell inhibitory receptor C-type lectin domain reveals the binding mode with N-glycan.

Human dendritic cell inhibitory receptor (DCIR) is a C-type lectin receptor expressed in classical dendritic cells and accepts several oligosaccharide ligands including N-glycans. Here, we report the crystal structures of human DCIR C-type lectin domains in the absence and presence of a branched N-glycan unit. The domain has a typical C-type lectin fold and two bound calcium ions. In the ligand-bound form, the disaccharide unit (GlcNAcß1-2Man) acceptably fits the electron density map, indicating that it forms the main epitope. The recognition of the nonterminal N-glycan unit explains the relatively broad specificity of this lectin.

Hypervalent iodine(III)-mediated oxidative decarboxylation of ß,γ-unsaturated carboxylic acids.

A novel oxidative decarboxylation of ß,γ-unsaturated carboxylic acids mediated by hypervalent iodine(III) reagents is described. The decarboxylative C-O bond forming reaction proceeded in the presence of PhI(OAc)2 to give the corresponding allylic acetates. In addition, decarboxylative C-N bond formation was achieved by utilizing hypervalent iodine(III) reagents containing an I-N bond. Mechanistic studies suggest the unique reactivity of hypervalent iodine reagents in this ionic oxidative decarboxylation.

The adipocyte-inducible secreted phospholipases PLA2G5 and PLA2G2E play distinct roles in obesity.

Metabolic disorders, including obesity and insulin resistance, have their basis in dysregulated lipid metabolism and low-grade inflammation. In a microarray search of unique lipase-related genes whose expressions are associated with obesity, we found that two secreted phospholipase A2s (sPLA2s), PLA2G5 and PLA2G2E, were robustly induced in adipocytes of obese mice. Analyses of Pla2g5(-/-) and Pla2g2e(-/-) mice revealed distinct roles of these sPLA2s in diet-induced obesity. PLA2G5 hydrolyzed phosphatidylcholine in fat-overladen low-density lipoprotein to release unsaturated fatty acids, which prevented palmitate-induced M1 macrophage polarization. As such, PLA2G5 tipped the immune balance toward an M2 state, thereby counteracting adipose tissue inflammation, insulin resistance, hyperlipidemia, and obesity. PLA2G2E altered minor lipoprotein phospholipids, phosphatidylserine and phosphatidylethanolamine, and moderately facilitated lipid accumulation in adipose tissue and liver. Collectively, the identification of "metabolic sPLA2s" adds this gene family to a growing list of lipolytic enzymes that act as metabolic coordinators.