Junji Kishino

Cell Adhesion to Phosphatidylserine Mediated by a Product of Growth Arrest-specific Gene 6

Gas6, a product of a growth arrest-specific gene 6, potentiates proliferation of vascular smooth muscle cells and prevents cell death of vascular smooth muscle cells. It has been also demonstrated that Gas6 is a ligand of receptor tyrosine kinases Axl, Sky, and Mer. Gas6 contains γ-carboxyglutamic acid residues, which are found in some blood coagulation factors and mediate the interaction of the coagulation factors with negatively charged phospholipid. In this study, we clarified that Gas6 specifically bound to phosphatidylserine and the binding was dependent on Ca2+ and γ-carboxyglutamic acid residues. Furthermore, we found that U937 cells, which express Gas6 receptor on their surfaces, adhered to phosphatidylserine-coated enzyme-linked immunosorbent assay (ELISA) plate only in the presence of Gas6 and Ca2+. U937 cells also bound to ELISA plate coated with phosphatidylinositol, but the binding was independent of Gas6 and Ca2+. On the other hand, U937 cells did not adhere to phosphatidylcholine- or phosphatidylethanolamine-coated ELISA plate even in the presence of Gas6 and Ca2+. These findings suggest that Gas6 may play a role in recognition of cells exposing phosphatidylserine on their surfaces by phagocytic cells, which is supposed to be one of the mechanisms for clearing dying cells.

Synthesis and Biological Activity of Various Derivatives of a Novel Class of Potent, Selective, and Orally Active Prostaglandin D2 Receptor Antagonists. 2. 6,6-Dimethylbicyclo[3.1.1]heptane Derivatives

In an earlier paper, we reported that novel prostaglandin D(2) (PGD(2)) receptor antagonists having the bicyclo[2.2.1]heptane ring system as a prostaglandin skeleton were a potent new class of antiallergic agents and suppressed various allergic inflammatory responses such as those observed in conjunctivitis and asthma models. In the present study, we synthesized PGD(2) receptor antagonists having the 6,6-dimethylbicyclo[3.1.1]heptane ring system. These derivatives have the amide moiety, in contrast to those with the bicyclo[2.2.1]heptane ring system, which have the sulfonamide group. The derivatives having the 6,6-dimethylbicyclo[3.1.1]heptane ring also exhibited strong activity in PGD(2) receptor binding and cAMP formation assays. In in vivo assays such as allergic rhinitis, conjunctivitis, and asthma models, these series of derivatives showed excellent pharmacological profiles. In particular, compound 45 also effectively suppressed eosinophil infiltration in allergic rhinitis and asthma models. This compound (45, S-5751) is now being developed as a promising alternative antiallergic drug candidate.

Contraction of guinea pig lung parenchyma by pancreatic type phospholipase A2 via its specific binding site

Porcine pancreatic group I phospholipase A2 (PLA2‐I) induced contraction of guinea pig lung parenchyma in a concentration‐dependent manner. Its EC50 value was similar to theK d value calculated from the specific binding of125I‐labeled porcine PLA2‐I in the membrane fraction of guinea pig lung. Type‐specific action of PLA2s and homologous desensitization strongly implicated the involvement of PLA2‐I‐specific sites in the activation process. Throm☐ane A2 was found to be the main product from lung tissue by PLA2‐I action and the contractile response by PLA2‐I was specifically suppressed by throm☐ane A2 receptor antagonists and cyclooxygenase inhibitor, but not by leukotriene receptor antagonist and H1 blocker. These findings indicate that PLA2‐I‐induced contractile response may depend on the secondarily produced throm☐ane A2, thus providing a new aspect of PLA2‐I from the pathophysiological standpoint.

Kinetic studies on stereospecific recognition by the thromboxane A2/prostaglandin H2 receptor of the antagonist, S-145.

1 The mechanism for the stereospecific recognition of the antagonist S‐145 by the thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor was examined by ligand‐binding techniques in rat vascular smooth muscle cells (VSMCs) and in human platelet membranes. 2 Scatchard analysis revealed the existence of a single class of binding sites with the same maximum number for both [3H]‐(+)‐S‐145 and [3H]‐(−)‐S‐145 in both cell types. The dissociation constants (Kd) for the binding of the (+)‐isomer in rat VSMCs and human platelet membranes were, respectively, 0.40 ± 0.03 and 0.20 ± 0.02 nm, each value being lower than that for the (−)‐isomer (3.57 ± 0.74 and 2.87 ± 0.08 nm, respectively). 3 The rank orders of potency (Ki) for a series of TXA2/PGH2 ligands at inhibiting [3H]‐(+)‐S‐145 binding were highly correlated with those determined for [3H]‐(−)‐S‐145 binding in both cell preparations. 4 Kinetic analysis of the binding of both radioligands revealed a much lower dissociation rate constant (k−1) and a slightly greater association rate constant (k1) for the (+)‐isomer compared to those for the (−)‐isomer. 5 These results suggest that it is at the stage of dissociation from the TXA2/PGH2 receptor that the stereochemistry of the optical isomers of S‐145 confers their difference in affinity for these receptors in rat VSMCs and human platelet membranes.

Novel binding sites of 15-deoxy-Δ12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.

Endothelin-induced intracellular Ca2+ mobilization through its specific receptors in murine peritoneal macrophages.

We studied the presence of specific binding sites for endothelin (ET) and the effect of ET on cytosolic free Ca2+ concentration ([Ca2+]i) in murine thiogylcolate‐activated peritoneal macrophages. Scatchard analysis for binding experiments using [125I]ET‐1 or [125I]ET‐3 revealed the existence of a single class of binding sites. The binding parameters (K??? and B???) for [125I]ET‐1 were almost identical to those for [125I]ET‐3. In addition, unlabeled 3 ET isopeptides (ET‐1, ET‐2 and ET‐3) inhibited the specific binding of both ET‐1 and ET‐3 with similar inhibitory potencies. All 3 ET isopeptides caused an increase in [Ca2+]i in the same dose‐dependent manner (0.01–100nM). These results demonstrate the existence of an ET receptor with the same affinity for all isoforms that mediates the ET‐induced intracellular Ca2+ mobilization in murine peritoneal macrophages.

Proteomic Identification of Protein Targets for 15-Deoxy-Δ12,14-Prostaglandin J2 in Neuronal Plasma Membrane

15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of factors contributed to the neurotoxicity of amyloid β (Aβ), a causative protein of Alzheimers disease. Type 2 receptor for prostaglandin D2 (DP2) and peroxysome-proliferator activated receptorγ (PPARγ) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ2, respectively. Previously, we reported that the cytotoxicity of 15d-PGJ2 was independent of DP2 and PPARγ, and suggested that 15d-PGJ2 induced apoptosis through the novel specific binding sites of 15d-PGJ2 different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ2 to amyloidoses, we performed binding assay [3H]15d-PGJ2 and specified targets for 15d-PGJ2 associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ2 and fibrillar Aβ. Specific binding sites of [3H]15d-PGJ2 were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [3H]15d-PGJ2 were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ2 in the plasma membrane. By using biotinylated 15d-PGJ2, eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ2 plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues.

Total Synthesis of Terprenin, a Highly Potent and Novel Immunoglobulin E Antibody Suppressant

Regioselective halogenations and Suzuki reactions ensure proper linkage of the aromatic rings in two total syntheses of terprenin (1). Both routes make it possible to prepare 1 efficiently and in large quantity.

Proliferative effect of phospholipase A2 in rat chondrocyte via its specific binding sites

We studied the presence of specific binding sites for pancreatic-type group I phospholipase A2 (PLA2-I), EC 3.1.1.4, and a PLA2-I action on the DNA synthesis of rat chondrocytes. Rat chondrocytes, derived from the xiphisternum of adult rats, had a single class of PLA2-I binding site with an equilibrium binding constant value of 0.9 nM and a maximum binding capacity of 53.9 fmol/10(6) cells. PLA2-I alone did not show any proliferative effect, however, PLA2-I dose-dependently stimulated thymidine incorporation in DNA in the presence of basic fibroblast growth factor (bFGF). The mammalian mature type of PLA2s-I specifically recognized the binding sites in these cells and had a synergistic effect on DNA synthesis with bFGF, whereas its inactive zymogen and group II PLA2 showed much lesser activities. The type-specific action of PLA2s implicated the involvement of PLA2-I specific binding sites in this activation process.

Receptor‐binding capability of pancreatic phospholipase A2 is separable from its enzymatic activity

Mammalian pancreatic phospholipase A2 (PLA2‐I) has its specific receptor through which PLA2‐I induces a variety of biological responses. In this study, a fundamental relationship between the enzymatic and the receptor‐binding activities of PLA2‐I was investigated. The specific binding of PLA2‐I to the receptor was found to be independent of Ca2+ which is requisite for the PLA2 activity. On the basis of this observation, we designed and produced mutant PLA2‐Is without Ca2+‐binding abilities in order to demonstrate that the structural requirement for the enzymatic activity of PLA2‐I is not identical with that for its receptor‐binding reaction. These mutant PLA2‐Is lost almost all enzymatic activity through a disturbance at the Ca2+‐binding site, as expected, but still retained a substantial affinity to the receptor, allowing us to conclude that the receptor‐binding reaction of PLA2‐I is separable from its catalytic action.