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The Journal of Antibiotics 06/2012; 65(9):483-5. · 1.65 Impact Factor
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ABSTRACT: SMTPs are a family of small-molecule plasminogen modulators that enhance plasminogen activation. SMTP-7, one of the most potent congeners, is effective in treating thrombotic cerebral infarction. The SMTP molecule consists of a tricyclic γ-lactam moiety, a geranylmethyl group, and an N-linked side chain. The presence of both an aromatic group and a negatively ionizable group in the N-linked side chain is crucial for activity. Investigations of the congeners with a phenylglycine-based side chain suggest that a phenolic hydroxy group affects potency. In this study, we isolate and characterize a series of novel SMTP congeners with a phenylamine-based N-linked side chain. Of the 11 congeners isolated, SMTP-19 (with a 4-phenylcarboxylic acid moiety), SMTP-22 (with a 3-hydroxyphenyl-4-carboxylic acid moiety) and SMTP-25 (with a 2-hydroxyphenyl-3-carboxylic acid moiety) are as potent as SMTP-7 in plasminogen-modulating activity. Their isomers with a carboxylic acid group and/or a phenolic hydroxy group at different positions have <40% of the activity of these congeners. Both SMTP-22 and SMTP-25 have >1.7 times more oxygen radical absorbance capacity as compared with SMTP-7.
The Journal of Antibiotics 04/2012; 65(7):361-7. · 1.65 Impact Factor
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ABSTRACT: Stachybotrys microspora triprenyl phenols (SMTPs) are a novel family of small molecules that enhance both activation and fibrin-binding of plasminogen. While their effects on fibrinolysis have been characterized in vitro, little is known about their activity in vivo with respect to plasminogen activation and blood clot clearance.
To select a potent SMTP congener for the evaluation of its action in vitro and in vivo, we tested several SMTP congeners with distinct structural properties for their effects on plasminogen activation. As a result, SMTP-7 (orniplabin) was found to have distinguished activity. Several lines of biochemical evidence supported the idea that SMTP-7 acted as a plasminogen modulator. SMTP-7 elevated plasma level of plasmin-α2-antiplasmin complex, an index of plasmin formation in vivo, 1.5-fold in mice after the intravenous injections at doses of 5 and 10 mg kg-1. In a rat pulmonary embolism model, SMTP-7 (5 mg kg-1) enhanced the rate of clot clearance ~3-fold in the absence of exogenous plasminogen activator. Clot clearance was enhanced further by 5 mg kg-1 of SMTP-7 in combination with single-chain urokinase-type plasminogen activator.
Our results show that SMTP-7 is a superior plasminogen modulator among the SMTP family compounds and suggest that the agent enhances plasmin generation in vivo, leading to clearance of thrombi in a model of pulmonary embolism.
Thrombosis Journal 01/2012; 10(1):2. · 1.31 Impact Factor
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The Journal of Antibiotics 11/2011; 65(2):91-3. · 1.65 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) formation has been found to induce the brain damage following stroke-like events. The aim of the present study was to investigate the effect of Stachybotrys microspora triprenyl phenol-7 (SMTP-7) on the generation of ROS in ischemia-induced cerebral infarction model and in vitro lipid peroxidation. We used immunohistochemistry and real-time reverse-transcription PCR for ex vivo evaluation and thiobarbituric acid-reactive substance reagent assay for in vitro evaluation. We demonstrated that SMTP-7 did not induce enhancement of 4-hydroxynonenal or neutrophil cytosolic factor 2 like t-PA administration at 3 h after ischemia ex vivo and reduce lipid peroxidation in vitro. This compound is the first low molecular weight compound with triplet activities of thrombolytic, anti-inflammatory, and antioxidant activities. We theorized that SMTP-7 is among the pharmacological agents that reduce ROS formation and have been found to limit the extent of brain damage following stroke-like events.
Archiv für Experimentelle Pathologie und Pharmakologie 07/2011; 384(1):103-8. · 2.65 Impact Factor
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ABSTRACT: Plasma hyaluronan-binding protein (PHBP), an activator of factor VII and prourokinase, is a serine protease circulating as a single-chain proenzyme (pro-PHBP). Pro-PHBP converts to the active two-chain form through autoproteolysis, and effectors that modulate autoactivation can regulate PHBP-mediated processes. Here, we show that histone promotes pro-PHBP autoactivation in vivo. Histone bound to pro-PHBP and promoted intermolecular pro-PHBP binding. Histone-mediated pro-PHBP activation in plasma leads to the formations of bradykinin and PHBP-α(2)-antiplasmin complex as well as histone degradation. Pro-PHBP activation was observed in the circulation of mice after injection of histone or lipopolysaccharide, which induced septic response accompanying extracellular histone release. Our results suggest pathophysiological relevance of histone-dependent pro-PHBP activation in hyperinflammatory process.
Biochemical and Biophysical Research Communications 06/2011; 409(3):483-8. · 2.48 Impact Factor
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ABSTRACT: A cDNA encoding a putative extracellular α-L-arabinofuranosidase was cloned from the basidiomycete Coprinopsis cinerea (CcAbf62A). CcAbf62A belongs to glycoside hydrolase family 62 (GH62), but is phylogenetically distinct from previously characterized GH62 enzymes. The recombinant CcAbf62A, expressed in Pichia pastoris, released L-arabinose from both wheat arabinoxylan and oat-spelt xylan. The enzyme activity for wheat arabinoxylan was increased by the addition of CcEst1, a carbohydrate esterase from C. cinerea.
Bioscience Biotechnology and Biochemistry 02/2011; 75(2):342-5. · 1.28 Impact Factor
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ABSTRACT: Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation.
The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice.
Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1β, TNF-α, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 μg/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions.
Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.
Stroke 02/2011; 42(4):1097-104. · 5.73 Impact Factor
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ABSTRACT: 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.
Bioscience Biotechnology and Biochemistry 01/2011; 75(10):2070-2. · 1.28 Impact Factor
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ABSTRACT: Malformin A₁, a cyclopentapeptide of fungal origin, enhances cellular fibrinolytic activity depending on the existence of a cofactor in blood plasma. However, the nature of this cofactor remains unknown. Here, we report that vitronectin acts as a plasma cofactor of malformin A₁. We purified the cofactor from bovine plasma by activity-based fractionation, and confirmed that vitronectin in conjunction with plasminogen supports the activity of malformin A₁ to promote the fibrinolytic activity of U937 cells. Malformin A₁ action was abolished by Arg-Gly-Asp peptide (a competitor of vitronectin-integrin binding), wortmannin (an inhibitor of signaling kinases), and cytochalasin B (an inhibitor of actin polymerization). Changes in actin organization and a decrease in filopodia were observed in cells treated with malformin A₁ and plasma. A focal localization of plasminogen on the cell surface was augmented by malformin A₁, whereas the amount of cell-surface-bound plasminogen was minimally altered by the treatment. Our results suggest the involvement of cytoskeletal reorganization via vitronectin signaling in the cellular fibrinolytic activity-enhancing action of malformin A₁.
Biological & Pharmaceutical Bulletin 01/2011; 34(9):1426-31. · 1.66 Impact Factor
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ABSTRACT: 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.
Biological & Pharmaceutical Bulletin 01/2011; 34(4):462-70. · 1.66 Impact Factor
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ABSTRACT: Plasma hyaluronan-binding protein (PHBP) is a serine protease implicated in proteolysis under inflammatory conditions. We identified laccaic acid, a widely used food coloring from scale insects, as a potent inhibitor of the protease in terms of both autoactivation of the PHBP proenzyme (IC(50) = 0.35-0.55 µg/ml) and the catalytic activity of the active enzyme (IC(50) = 1.1 µg/ml).
Bioscience Biotechnology and Biochemistry 11/2010; 74(11):2320-2. · 1.28 Impact Factor
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ABSTRACT: 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.
Thrombosis Research 11/2010; 126(5):406-13. · 2.44 Impact Factor
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ABSTRACT: The fungal metabolite Stachybotrys microspora triprenyl phenols (SMTPs) are small-molecule plasminogen modulators that enhance plasminogen activation. The SMTP molecule consists of a tricyclic γ-lactam moiety, an isoprene side-chain and an N-linked side-chain. Previous investigations have demonstrated that the N-linked side-chain is crucial for its activity. In this study, we have isolated 11 new SMTP congeners with a variety of N-linked side-chain structures, to investigate structure-activity relationships. Active compounds included congeners with a carboxyl or a sulfonic acid group in the N-linked side-chain, whereas not all the congeners with a carboxyl group were active. Of these congeners, that with methionine or tyrosine as the N-linked side-chain moiety was more active than that with an aliphatic amino acid. Congeners without ionizable group in the N-linked side-chain were essentially inactive.
The Journal of Antibiotics 10/2010; 63(10):589-93. · 1.65 Impact Factor
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ABSTRACT: The coagulation and fibrinolytic systems are central to the hemostatic mechanism, which works promptly on vascular injury and tissue damage. The rapid response is generated by specific molecular interactions between components in these systems. Thus, the regulation mechanism of the systems is programmed in each component, as exemplified by the elegant processes in zymogen activation. This review describes recently identified small molecules that modulate the activation of zymogens in the fibrinolytic and coagulation systems.
FEBS Journal 09/2010; 277(18):3675-87. · 3.79 Impact Factor
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ABSTRACT: Tissue plasminogen activator (t-PA) has a short therapeutic time window for administration (3 h) and carries a risk of promoting intracerebral hemorrhage. The aim of the present study was to investigate a therapeutic time window and frequency of hemorrhagic region by treatment with Stachybotrys microspora triprenyl phenol-7 (SMTP-7). Thrombotic occlusion was induced by transfer of acetic acid-induced thrombus at the right common carotid artery into the brain of mice. Infarction area, neurological score, edema percentage, and regional cerebral blood flow (CBF) were determined as the index of the efficacy of SMTP-7. In order to evaluate the mechanism of SMTP-7, plasmin activities and the expressions of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and IL-6 mRNA were examined. SMTP-7 (0.1, 1, 10 mg/kg) dose dependently reduced infarction area, neurological score, and edema percentage. Additionally, its therapeutic time window was longer than that of t-PA, a high-molecular-weight compound. In addition, little hemorrhagic region was induced by treatment with SMTP-7. SMTP-7 showed plasmin activity in vivo and caused a decreased CBF to recover. Furthermore, the expressions of inflammatory cytokine mRNA (IL-1beta, TNF-alpha, IL-6) were increased by t-PA treatment 3 h after ischemia but were not induced by SMTP-7 treatment. These results indicate that SMTP-7 shows potential thrombolytic and anti-inflammatory effects as well as a wide therapeutic time window and little hemorrhagic region compared with that of t-PA. Therefore, this novel low-molecular-weight compound may represent a novel approach for the treatment of cerebral infarction.
Archiv für Experimentelle Pathologie und Pharmakologie 09/2010; 382(3):245-53. · 2.65 Impact Factor
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ABSTRACT: Mulberry leaves are rich in 1-deoxynojirimycin (DNJ), an inhibitor of α-glucosidase. We previously showed that DNJ-rich mulberry leaf extract suppressed elevation of postprandial blood glucose in humans. The objective of this study was to evaluate the effects of DNJ-rich mulberry leaf extract on plasma lipid profiles in humans. An open-label, single-group study was conducted in 10 subjects with initial serum triglyceride (TG) level ≥200 mg/dl. Subjects ingested capsules containing DNJ-rich mulberry leaf extract at 12 mg three times daily before meals for 12 weeks. Our findings showed a modest decrease in serum TG level and beneficial changes in the lipoprotein profile following 12-week administration of DNJ-rich mulberry leaf extract. No significant changes in hematological or biochemical parameters were observed during the study period; no adverse events associated with DNJ-rich mulberry leaf extract occurred.
Journal of Clinical Biochemistry and Nutrition 09/2010; 47(2):155-61. · 1.98 Impact Factor
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ABSTRACT: The aim of the present study was to establish a novel embolic model of cerebral infarction and to evaluate the effect of Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a novel fungal triprenyl phenol metabolite. Thrombotic occlusion was induced by transfer of acetic acid-induced embolus into the brain. The regional cerebral blood flow was measured by a laser Doppler flowmeter to check the ischemic condition. Infarction area was assessed by 2% 2,3,5-triphenyltetrazolium chloride (TTC) staining. Neurological scores were determined by a modified version of the method described by Longa et al. Emboli were accumulated at the temporal or parietal region of the middle cerebral artery. Additionally, we found that this model showed decreased cerebral blood flow and increased infarction area and neurological scores. Treatment with tissue plasminogen activator (t-PA) reduced infarction area and the neurological scores in a dose-dependent manner; moreover, the decreased cerebral blood flow recovered. SMTP-7 also reduced these values. The therapeutic time window of SMTP-7 was longer than that of t-PA. These results indicate that this model may be useful for understanding the pathophysiological mechanisms of cerebral infarction and evaluating the effects of therapeutic agents. Additionally, SMTP-7 is a promising approach to extend the therapeutic time window. Therefore, this novel compound may represent a novel approach for the treatment of cerebral infarction.
Journal of Pharmacological Sciences 01/2010; 114(1):41-9. · 2.08 Impact Factor
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ABSTRACT: 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.
Biological & Pharmaceutical Bulletin 01/2010; 33(8):1430-3. · 1.66 Impact Factor
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ABSTRACT: Plactin, a family of cyclopentapeptides, enhances fibrinolytic activity by elevating the activity of cellular urokinase-type plasminogen activator (u-PA), a protease involved in a variety of extracellular proteolytic events. Factor(s) in the blood plasma is an absolute requirement for this plactin activity. In this study, we found that plactin promoted plasma cofactor-dependent conversion of inactive single-chain u-PA to active two-chain u-PA on U937 cells. Using plactin-affinity chromatography, we identified prothrombin as one of the plasma cofactors. In incubations of U937 cells with prothrombin and Xa, plactin increased the formation of thrombin, which cleaved single-chain u-PA to afford the inactive two-chain form. Thrombin-cleaved two-chain u-PA was alternatively activated by cellular cystatin-sensitive peptidase activity, yielding fully active two-chain u-PA. In a purified system, plactin bound to prothrombin, altered its conformation and dually modulated factor Xa-mediated proteolytic activation of prothrombin to alpha-thrombin. Plactin inhibited the activation catalyzed by Xa in complex with Va, Ca(2+) and phospholipids (prothrombinase), whereas the activations catalyzed by nonmembrane-associated Xa were enhanced markedly by plactin. Plactin inhibited in vitro plasma coagulation, which involved prothrombinase formation. Plactin did not cause prothrombin activation or thrombosis in normal mice at doses that produced a protective effect in a thrombin-induced pulmonary embolism mouse model. Therefore, the dual modulation of prothrombin activation by plactin may be interpreted as leading to anticoagulation under physiological coagulating conditions.
FEBS Journal 06/2009; 276(9):2516-28. · 3.79 Impact Factor
