Alprostadil suppresses angiogenesis in vitro and in vivo in the murine Matrigel plug assay.
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1. Prostaglandin E(1) (PGE(1), alprostadil) is used as a vasodilator for the treatment of peripheral vascular diseases. 2. Previous reports suggested a pro-angiogenic effect for PGE(1). 3. We studied the in vitro and in vivo effect of PGE(1), complexed with alpha-cyclodextrin, on the angiogenic process. Contrary to what was expected, we found that, in human umbilical vein endothelial cells (HUVECs), PGE(1) inhibited proliferation, migration and capillary-like structure formation in Matrigel. 4. By RT-PCR studies, the expression of the EP(2) and EP(3) subtypes of the PG receptor was detected in HUVECs. 5. PGE(1) alone stimulated adenylate cyclase activity at micromolar concentrations, while at nanomolar concentrations potentiated the forskolin-induced cAMP accumulation. 6. 8-Bromoadenosine-3':5'-cyclic monophosphate (Br-cAMP) mimicked the inhibitory effect of PGE(1) on endothelial cell growth, motility and tube formation. 7. Sulprostone, an agonist at the EP(3) subtype of PG receptors, mimicked the in vitro anti-angiogenic effects of PGE(1), while butaprost, an EP(2) receptor agonist, had no effect. 8. Finally, in the plug assay model of angiogenesis in mice, PGE(1) showed a strong inhibitory effect on Matrigel neovascularization. 9. Thus, PGE(1) possesses strong anti-angiogenic activity in vitro and in vivo. (+info)
The binding of beta- and gamma-cyclodextrins to glycogen phosphorylase b: kinetic and crystallographic studies.
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A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, alpha-, beta-, and gamma-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with K(i) values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with beta- and gamma-cyclodextrins at 1.94 A and 2.3 A resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that beta- and gamma-cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of alpha-cyclodextrin and octakis (2,3,6-tri-O-methyl)-gamma-cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction. (+info)
Encapsulation of shiitake (Lenthinus edodes) flavors by spray drying.
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Powdery encapsulation of shiitake flavors, extracted from dried shiitake, was investigated by spray drying. Flavor retention increased with an increase in drying air temperature and solid content, and decreased with an increase in dextrose equivalents of maltodextrin. A heat-treatment of the extract liquid made the lenthionine concentration increase, but did not influence the concentrations of the other flavors. The formation of lenthionine with heat-treatment could be described by the consecutive unimolecular-type first order reaction. Lenthionine content in a spray-dried powder prepared with the heated extracted liquid significantly increased. alpha-Cyclodextrin was the most suitable encapsulant of alpha-, beta-, and gamma-cyclodextrins to prepare the spray-dried powder, including lenthionine. The flavor retentions were markedly increased by using of alpha-cyclodextrin and maltodextrin in combination as an encapsulant. (+info)
A modified method using static head-space gas chromatography for determining the stability constants of 1-alkanol/alpha-cyclodextrin complexation.
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A modification of the conventional static head-space gas chromatography method (SHSGC method) to determine stability constants for 1-alkanol/alpha-CD inclusion complexes was investigated. The 1 : 1 stability constants determined by this modified SHSGC method are in reasonable agreement with the corresponding values reported previously. The modified SHSGC method precludes the necessity of the calibration curve by the use of Henry's law constant of guest. Consequently, the modified SHSGC method is more advantageous than the conventional SHSGC method because the experimental time required for determination of the stability constant is markedly reduced. (+info)
Three-dimensional structure of soybean beta-amylase determined at 3.0 A resolution: preliminary chain tracing of the complex with alpha-cyclodextrin.
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The three-dimensional structure of a complex of soybean beta-amylase [EC 3.2.1.2] with an inhibitor, alpha-cyclodextrin, has been determined at 3.0 A resolution by X-ray diffraction analysis. Preliminary chain tracing showed that the enzyme folded into large and small domains. The large domain has a (beta alpha)8 super-secondary structure, while the smaller one is formed from two long loops extending from the beta 3 and beta 4 strands of the (beta alpha)8 structure. The interface of the two domains together with shorter loops from the (beta alpha)8 structure form a deep cleft, in which alpha-cyclodextrin binds slightly away from the center. Two maltose molecules also bind in the cleft. One shares a binding site with alpha-cyclodextrin and the other is situated more deeply in the cleft. (+info)
Inhibitory effect of OP-41483.alpha-CD, a prostacyclin analog, on peripheral vascular lesion models in rats.
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The effect of a chemically stable prostacyclin analog, OP-41483 alpha-cyclodextrin clathrate (OP-41483.alpha-CD), on vascular lesions, platelet aggregation and blood pressure were examined and compared with those of prostaglandin E1 alpha-cyclodextrin clathrate (PGE1.CD) in in vivo rat models. 1) In the laurate (1 mg/leg, i.a.)-induced arterial thrombotic model, OP-41483.alpha-CD (1 microgram/kg/min, i.v.) prevented the progression of femoral arterial vascular lesions and enhanced the development of collaterals in the femoral artery. PGE1.CD did not inhibit the progression of vascular damages. 2) In the model of vasoconstriction induced by epinephrine (0.05 mg/tail, s.c.) and ergotamine (2 mg/kg, s.c.), OP-41483.alpha-CD and PGE1.CD, at 1 microgram/kg/min, inhibited the progress of of tail gangrene and lessened the decrease in tail cutaneous blood flow. 3) OP-41483.alpha-CD (1 microgram/kg/min) suppressed the ADP (0.1 mg/kg/min, i.v.)-induced decrease in the number of circulating platelets without affecting the change in blood pressure. In contrast, PGE1.CD (3 micrograms/kg/min) inhibited ADP-induced thrombocytopenia with a decrease in blood pressure. These results indicate that OP-41483.alpha-CD has antiplatelet and cutaneous blood flow improving activities that are greater than its hypotensive effect and may be of therapeutic potential in peripheral vascular diseases. (+info)
Behavior of alpha-, beta-, and gamma-cyclodextrins and their derivatives on an in vitro model of blood-brain barrier.
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Cyclodextrins (CDs) can be envisaged to cure some diseases related to the brain, but the behavior of these compounds toward the blood-brain barrier (BBB) remains largely unexplored to envisage such clinical applications. To fulfill this gap, the toxicity and endothelial permeability for native, methylated, and hydroxypropylated alpha-, beta-, and gamma-CDs have been studied on an in vitro model of BBB. As shown by the endothelial permeability for sucrose and immunofluorescence stainings, the native CDs are the most toxic CDs (alpha- > beta- > gamma-CD). Whereas the chemical modification of beta-CD did not affect the toxicity of this CD, differences are observed for the alpha- and gamma-CD. To determine the origin of toxicity, lipid effluxes on the brain capillary endothelial cells were performed in the presence of native CDs. It was found that alpha-CD removed phospholipids and that beta-CD extracted phospholipids and cholesterol. gamma-CD was less lipid-selective than the other CDs. Finally, the endothelial permeability of each CD has been determined. Surprisingly, no structure/permeability relationship has been observed according to the nature and chemical modifications of CDs. (+info)
Two additional carbohydrate-binding sites of beta-amylase from Bacillus cereus var. mycoides are involved in hydrolysis and raw starch-binding.
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In the previous X-ray crystallographic study, it was found that beta-amylase from Bacillus cereus var. mycoides has three carbohydrate-binding sites aside from the active site: two (Site2 and Site3) in domain B and one (Site1) in domain C. To investigate the roles of these sites in the catalytic reaction and raw starch-binding, Site1 and Site2 were mutated. From analyses of the raw starch-binding of wild-type and mutant enzymes, it was found that Site1 contributes to the binding affinity to raw-starch more than Site2, and that the binding capacity is maintained when either Site1 or Site2 exists. The raw starch-digesting ability of this enzyme was poor. From inhibition studies by maltitol, GGX and alpha-CD for hydrolyses of maltopentaose (G5) and amylose ( (n) = 16) catalyzed by wild-type and mutant enzymes, it was found that alpha-CD is a competitive inhibitor, while, maltitol behaves as a mixed-type or competitive inhibitor depending on the chain length of the substrate and the mutant enzyme. From the analysis of the inhibition mechanism, we conclude that the bindings of maltitol and GGX to Site2 in domain B form an abortive ESI complex when amylose ( (n) = 16) is used as a substrate. (+info)