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.

To optimize a receptor-mediated and cell-selective gene transfer with polyethyleneimine (PEI)-based vector, we synthesized three galactosylated PEIs (Gal-PEI) with different molecular weights (PEI(1800), PEI(10,000), and PEI(70,000)) and investigated their potential as a targetable vector to asialoglycoprotein receptor-positive cells. All PEI derivatives formed complexes with plasmid DNA (pDNA), whereas the particle size of the complex became smaller on increasing the molecular weight of PEI. Transfection efficiency in HepG2 cells with PEI was highest with PEI(1800); efficiency was next highest with PEI(10,000), although the cellular association was similar. After galactosylation, Gal(19)-PEI(10,000)/pDNA and Gal(120)-PEI(70,000)/pDNA showed considerable agglutination with a galactose-recognizing lectin, but Gal(9)-PEI(1800) did not, suggesting that galactose units on the Gal(9)-PEI(1800)-pDNA complex are not sufficiently available for recognition. Gal(19)-PEI(10,000)-pDNA and Gal(120)-PEI(70,000)-pDNA complexes showed galactose-inhibitable transgene expression in HepG2 cells. Transfection efficiency was greatest with Gal(19)-PEI(10,000)/pDNA, a result that highlights the importance of obtaining a balance between the cytotoxicity and the transfection activity, both of which are found to be a function of the molecular weight of PEI. After intraportal injection, however, Gal(153)-PEI(70,000)/pDNA having a low N/P ratio was most effective, suggesting that additional variables, such as the size of the complex, are important for in vivo gene transfer to hepatocytes.

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PGE2 has long been known as a potentiator of acute inflammation, but its mechanisms of action still remain to be defined. In this study, we employed inflammatory swelling induced in mice by arachidonate and PGE2 as models and dissected the role and mechanisms of action of each EP receptor at the molecular level. Arachidonate- or PGE2-induced vascular permeability was significantly reduced in EP3-deficient mice. Intriguingly, the PGE2-induced response was suppressed by histamine H1 antagonist treatment, histidine decarboxylase deficiency, and mast cell deficiency. The impaired PGE2-induced response in mast cell–deficient mice was rescued upon reconstitution with wild-type mast cells but not with EP3-deficient mast cells. Although the number of mast cells, protease activity, and histamine contents in ear tissues in EP3-deficient mice were comparable to those in wild-type mice, the histamine contents in ear tissues were attenuated upon PGE2 treatment in wild-type but not in EP3-deficient mice. Consistently, PGE2–EP3 signaling elicited histamine release in mouse peritoneal and bone marrow–derived mast cells, and it exerted degranulation and IL-6 production in a manner sensitive to pertussis toxin and a PI3K inhibitor and dependent on extracellular Ca2+ ions. These results demonstrate that PGE2 triggers mast cell activation via an EP3–Gi/o–Ca2+ influx/PI3K pathway, and this mechanism underlies PGE2-induced vascular permeability and consequent edema formation.

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G protein-coupled receptors (GPCRs), which are indispensable to life and also implicated in a number of diseases, construct important drug targets. For the efficient structure-guided drug design, however, their structural stabilities must be enhanced. An amino-acid mutation is known to possibly lead to the enhancement, but currently available experimental and theoretical methods for identifying stabilizing mutations suffer such drawbacks as the incapability of exploring the whole mutational space with minor effort and the unambiguous physical origin of the enhanced or lowered stability. In general, after the identification is successfully made for a GPCR, the whole procedure must be followed all over again for the identification for another GPCR. Here we report a theoretical strategy by which many different GPCRs can be considered at the same time. The strategy is illustrated for three GPCRs of Class A in the inactive state. We argue that a mutation of the residue at a position of N = 3.39 (N is the Ballesteros-Weinstein number), a hot-spot residue, leads to substantially higher stability for significantly many GPCRs of Class A in the inactive state. The most stabilizing mutations of the residues with N = 3.39 are then identified for two of the three GPCRs, using the improved version of our free-energy function. These identifications are experimentally corroborated, which is followed by the determination of new three-dimensional (3D) structures for the two GPCRs. We expect that on the basis of the strategy, the 3D structures of many GPCRs of Class A can be solved for the first time in succession.

Adipogenesis is a crucial aspect in controlling body fat mass ( 1, 2 ). Acquisition of the mature adipocyte phenotype is a highly regulated process in which mesenchymal stem cells (MSC) undergo differentiation, resulting in both an increase in size and number of mature adipocytes in adipose tissue. Adipose tissue is important not only for energy storage but also as an endocrine organ that regulates energy homeostasis by secreting various adipokines, such as cytokines, chemokines, growth factors, and lipid mediators ( 3 ). The presence of receptors for adipokines in preadipocytes and adipocytes has been shown, suggesting that secreted adipokines have autocrine effects and regulate their own differentiation and functions ( 4 ). Although it has been shown that a number of factors, including adipokines, regulate adipogenesis in various settings, most of the evidence comes from supraphysiological or pharmacological doses of these molecules to elicit a response. Hence, their physiological signifi cance in local milieu has not been established.Prostaglandins (PG) are arachidonate metabolites synthesized by the action of cyclooxygenase (COX) as the rate-limiting enzyme. COX has been shown to exist as two isomers, COX-1 and COX-2. PGs exert a wide range of actions through their binding to plasma membrane receptors ( 5, 6 ). For instance, PGF 2 ␣ exerts its actions via specifi c interactions with the prostanoid FP receptor, which activates phopholipase C, resulting in phophatidylinositol breakdown ( 7 ). In contrast, PGE 2 exerts action through its Abstract The prostaglandin (PG) receptors EP4 and FP have the potential to exert negative effects on adipogenesis, but the exact contribution of endogenous PG-driven receptor signaling to this process is not fully understood. In this study, we employed an adipocyte differentiation system from mouse embryonic fi broblasts (MEF) and compared the effects of each PG receptor-defi ciency on adipocyte differentiation.