Vulgatibacter incomptus gen. nov., sp. nov. and Labilithrix luteola gen. nov., sp. nov., two myxobacteria isolated from soil in Yakushima Island, and the description of Vulgatibacteraceae fam. nov., Labilitrichaceae fam. nov. and Anaeromyxobacteraceae fam. nov.

Two myxobacterial strains (designated B00001(T) and B00002(T)) were isolated from forest soil samples collected from Yakushima Island, Kagoshima, Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains B00001(T) and B00002(T) respectively formed independent branches within the suborders Cystobacterineae and Sorangiineae and were most closely related to Cystobacter armeniaca DSM 14710(T) (90.4% similarity) and Byssovorax cruenta DSM 14553(T) (91.3%). Neither strain showed typical features of myxobacteria such as bacteriolytic action or fruiting body formation, but both had high DNA G+C contents (66.3-68.3 mol%). Swarming motility was observed in strain B00002(T) only. Cells of both strains were vegetative, chemoheterotrophic, mesophilic, strictly aerobic, Gram-negative, motile rods, and both strains exhibited esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and ß-galactosidase activities. Strain B00001(T) contained MK-7 as the predominant respiratory quinone and the major fatty acid was iso-C15:0. In contrast, strain B00002(T) contained MK-8 as the major cellular quinone and the major fatty acids were C16 : 1ω5c and iso-C17 : 0. Based on the phenotypic and genotypic data presented, strains B00001(T) and B00002(T) represent novel genera and species, for which we propose the names Vulgatibacter incomptus gen. nov., sp. nov. and Labilithrix luteola gen. nov., sp. nov., respectively. The type strains of Vulgatibacter incomptus and Labilithrix luteola are B00001(T) ( = NBRC 109945(T) = DSM 27710(T)) and B00002(T) ( = NBRC 109946(T) = DSM 27648(T)), respectively. The new genera are assigned to the new families Vulgatibacteraceae fam. nov. and Labilitrichaceae fam. nov., respectively. In addition, Anaeromyxobacteraceae fam. nov., is proposed to accommodate the genus Anaeromyxobacter, which is related to the genus Vulgatibacter.

Elucidation of crucial structures for a catechol-based inhibitor of plasma hyaluronan-binding protein (factor VII activating protease) autoactivation.

Plasma hyaluronan-binding protein (PHBP) is a serine protease the activation of which is implicated in inflammation. Previous investigations have suggested the presence of catechol-binding sites in its proenzyme form, pro-PHBP. Here we found that compounds with plural catechol groups conjugated with strong electron-withdrawing groups, such as tyrphostin AG 537 (IC(50)=18 nM), were potent inhibitors of pro-PHBP activation.

Inhibitors of autoactivation of plasma hyaluronan-binding protein (factor VII activating protease).

Plasma hyaluronan-binding protein (PHBP), a serine protease that can activate coagulation factor VII and prourokinase, circulates in a single-chain form (pro-PHBP) and autoproteolytically converts to an active two-chain form with the aid of an effector such as spermidine and heparin. It has been postulated that PHBP plays roles in regulating inflammation, vascular function, fibrosis and atherosclerosis. From the comprehensive screening of natural sources for inhibitors of spermidine-induced pro-PHBP autoactivation, we identified several compounds with a polyphenol feature. Of these inhibitors, tannic acid (IC(50)=0.020 µM), delphinidin (IC(50)=0.079 µM), hamamelitannin (IC(50)=0.19 µM), (-)-epicatechin gallate (IC(50)=0.24 µM), and 3,5-di-O-caffeoylquinic acid (IC(50)=1.0 µM) were potent and selective, and did not inhibit heparin-induced pro-PHBP autoactivation and the active form of PHBP at concentrations 100 times higher than the respective IC(50) values. From evaluation of the activities of related compounds, it has been suggested that a compound with multiple aromatic rings with plural phenolic hydroxyl substituents exhibits potent activity. The inhibitory actions of delphinidin, hamamelitannin, (-)-epicatechin gallate and 3,5-di-O-caffeoylquinic acid were attenuated by catechol, a minimum polyphenol unit. Thus, it is likely that pro-PHBP binds these potent inhibitors through its site(s) that recognize a catechol-like structure. Our results would facilitate understanding of the molecular mechanism of pro-PHBP autoactivation and rational design of a compound for suppressing unregulated pro-PHBP activation.

The cyclopentapeptide plactin enhances cellular binding and autoactivation of the serine protease plasma hyaluronan-binding protein.

Plactin, a family of cyclopentapeptides of fungal origin, enhances fibrinolytic activity by promoting of single-chain urokinase-type plasminogen activator (scu-PA) activation on the cell surface. For this activity, factor(s) in the blood plasma is absolutely required. In the previous studies, we identified prothrombin as a plasma cofactor involved in this mechanism, while the presence of another independent cofactor was suggested. The objective of this study was to identify the second cofactor and investigate the mechanism involved. Using plactin-affinity and ion-exchange chromatographies, we purified plasma hyaluronan-binding protein (PHBP) ~4,000-fold from human plasma as an independent plactin cofactor. PHBP, at ~10nM, was effective in plactin-dependent promotion of scu-PA activation by U937 cells. PHBP is a serine protease that is produced as a single-chain proenzyme (pro-PHBP) and autoproteolytically converted to an active two-chain form. Pro-PHBP was comparable to PHBP in activity to promote plactin-dependent scu-PA activation by U937 cells. Plactin enhanced both cellular binding and autoproteolytic activation of pro-PHBP. The two activities were obtained with a plactin concentration at ~30µM, which resulted in a significant increase in intrinsic fluorescence and self association of pro-PHBP. Thus, it is suggested that such changes account for both enhanced cellular binding and autoactivation of pro-PHBP, resulting in an enhancement of scu-PA activation.

Purpurin as a specific inhibitor of spermidine-induced autoactivation of the protease plasma hyaluronan-binding protein.

Plasma hyaluronan-binding protein (PHBP), a serine protease that can activate coagulation factor VII and prourokinase, circulates as a single-chain form (pro-PHBP), and is autoproteolytically converted to an active two-chain form with the aid of an effector such as spermidine and heparin. In this study, we screened natural sources for inhibitors of spermidine-induced pro-PHBP autoactivation. As an active agent, we purified bikaverin from a culture of a fungus. Bikaverin inhibited spermidine-induced autoactivation with an IC(50) of 0.45 microM, while it also inhibited the active form of PHBP (IC(50)=0.8 microM). Additional screening of related compounds led to the identification of purpurin, a plant anthraquinone, as a specific inhibitor: IC(50)=6.6 microM for spermidine-induced autoactivation; no inhibition of heparin-induced autoactivation and active PHBP. Alizarin and emodin, which structurally differed from purpurin in the position or the number of the hydroxyl groups, were less active and nonspecific. Thus, the position and/or the number of the hydroxyl group affect both the potency and selectivity of the anthraquinone inhibitors.

Optimization of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives as arginine vasopressin V2 receptor agonists and discussion of their binding modes.

A series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives were optimized to achieve potent agonistic activity, both in vitro and in vivo, for the arginine vasopressin V(2) receptor, resulting in the eventual discovery of compound 1g. Molecular modeling of compound 1g with V(2) receptor was also examined to evaluate the binding mode of this series of compounds.

Synthesis and structure-activity relationships of amide derivatives of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetic acid as selective arginine vasopressin V2 receptor agonists.

A series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetamide derivatives was synthesized, and their structure-activity relationships were examined in order to identify potent and selective arginine vasopressin V(2) receptor agonists. Attempts to substitute other chemical groups in place of the 2-pyridilmethyl moiety of 1a led to the discovery that potent V(2) binding affinity could be obtained with a wide range of functional groups. This structural tolerance allowed for the manipulation of other attributes, such as selectivity against V(1a) receptor affinity or avoidance of the undesirable inhibition of cytochrome P450 (CYP), without losing potent affinity for the V(2) receptor. Some representative compounds obtained in this study were also found to decrease urine volume in awake rats.

Preparation of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetamide derivatives as novel arginine vasopressin V(2) receptor agonists.

The present work describes the discovery of novel series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives as arginine vasopressin (AVP) V(2) receptor agonists. By replacing the amide juncture in YM-35278 with a direct ring connection gave compound 10a, which acts as a V(2) receptor agonist. These studies provided the potent, orally active non-peptidic V(2) receptor agonists 10a and 10j.

Effect of YM-254890, a specific Galphaq/11 inhibitor, on experimental peripheral arterial disease in rats.

The protective effect of YM-254890, a specific Galphaq/11 inhibitor, on laurate-induced peripheral arterial disease in rats was compared with those of prostaglandin E1 (PGE1), beraprost, and clopidogrel. YM-254890 inhibited ADP-induced ex vivo rat platelet aggregation at a dose of 3 microg/kg. Furthermore, YM-254890 strongly inhibited phenylephrine-, serotonin- and endothelin-1-induced contractions in the rat aorta, and improved dermal blood flow after the laurate injection. The intra-arterial single bolus administration of YM-254890 15 min after the laurate injection dose-dependently inhibited the progression of the lesion, with significance, at 3 microg/kg without affecting systemic blood pressure. PGE1 and beraprost, when administered before the laurate injection, were effective, but their potencies were less than that of YM-254890. Clopidogrel significantly suppressed lesion progression when administered at 30 mg/kg twice a day for 3 days, which completely inhibited platelet aggregation. These results suggest that the local administration of YM-254890 may be useful for treating peripheral arterial disease.