Akira Ogamo

Sulfated Polysaccharides Enhance the Biological Activities of Bone Morphogenetic Proteins

Bone morphogenetic proteins (BMPs), which have been shown to be heparin-binding proteins, induce osteoblast differentiation in mesenchymal cells. In the present study, we examined the effects of heparin on the BMP activities in C2C12 myoblasts. Heparin dose dependently enhanced the osteoblast differentiation induced by not only homodimers of BMP-2 or BMP-4 but also heterodimers of BMP-2/6 or BMP-2/7. However, the osteoblast differentiation induced by the constitutively active BMPR-IA, a functional BMP type I receptor, was not affected by heparin. Heparan sulfate and dextran sulfate also enhanced the BMP-2 activity, although the chemically desulfated heparin-derivatives have lost this stimulatory capacity. Heparin dose-dependently suppressed the accumulation of BMP-2 from the culture media into the cell layer or BMPR-IA, and retained a large amount of BMP-2 in the culture media. The biological activity of BMP-2, which was evaluated using a BMP-responsive reporter gene expression, was prolonged in the presence of heparin. Taken together, these results suggest that sulfated polysaccharides enhance the biological activity of both homodimers and heterodimers of BMPs by continuously serving the ligands to their signaling receptors expressed on cell membranes.

Preparation and anti-HIV activity of low-molecular-weight carrageenans and their sulfated derivatives

Abstract Lambda-, kappa-, and iota-carrageenan were depolymerized in the presence of ferrous ions or ferrous ions plus ascorbic acid at room temperature. Tetrabutyl-ammonium salts of lambda-, kappa- and iota-carrageenan were dissolved in N,N -dimethylformamide and sulfated by the addition of pyridine-sulfur trioxide in N,N -dimethylformamide. Then, the sulfated carrageenans were depolymerized by the incubation with ferrous ions and ascorbic acid. The anti-HIV activities of the various preparations were determined in a system in which MT-4 cells were infected with HTLV-IIIB. The activity of lambda-carrageenan was similar to that of dextran sulfate and the depolymerized lambda-carrageenan retained high activity. The activities of kappa- and iota-carrageenan and their depolymerized forms were about 6% of that of lambda-carrageenan. The sulfation of kappa-and iota-carrageenan increased the activities of these compounds to the same level as that of lambda-carrageenan. However, the sulfation of lambda-carrageenan did not affect its activity. Depolymerized sulfated kappa- and iota-carrageenan with mean molecular weights of 50,000 (81,000-46,000) had higher anti-HIV activity than dextran sulfate and all other preparations of carrageenan tested.

Preparation and properties of fluorescent glycosamino-glycuronans labeled with 5-aminofluorescein

The uronic acid residues of all known glycosaminoglycuronans reacted with 5-aminofluorescein to yield fluorescent glycosaminoglycuronan derivatives, which showed fluorescence characteristics identical to those of fluorescein or 5-acetamidofluorescein. The fluorescent products could be purified by chromatography on Octyl-Sepharose; three preparations of labeled chondroitin 6-sulfate having different degrees of substitution, and a labeled heparin were obtained. Fluorescent hyaluronic acid containing labeled and unlabeled molecules was digested with testicular hyaluronidase to give fluorescent oligosaccharides. Fluorescent chondroitin 6-sulfate was treated with chondroitinase AC to give a nonfluorescent disaccharide and minor proportion of fluorescent octasaccharide. Fluorescent heparin retained its anticoagulant activity, which was similar to that of the starting heparin; its half-life in circulating rabbit blood was 36 min (by fluorometry) and 45 min (by clotting-time assay).

Binding of heparin fractions and other polysulfated polysaccharides to plasma fibronectin: effects of molecular size and degree of sulfation of polysaccharides.

Heparin was divided into four fractions on fibronectin-Sepharose. The higher affinity fraction for fibronectin was larger in molecular size, higher in sulfate content and higher in affinity for anti-thrombin III. Together with these heparin fractions, the following three series of heparin samples were examined to compare the affinity for fibronectin-Sepharose: four fractions separated on Sephadex G-100; five fractions separated on antithrombin III-Sepharose, and six partially and completely N-desulfated heparins. The result showed that the affinity of heparin for fibronectin was dependent exclusively on its molecular size, and that an appropriate level of sulfate content in heparin (1.9-2.4 mol/disaccharide) was essential for the affinity. The sulfated preparations of glycosaminoglycans (heparan sulfate, dermatan sulfate and chondroitin 4-sulfate) and neutral polysaccharides (amylose and dextran) having higher sulfate content than heparin were found to display higher affinity for fibronectin than heparin. This suggested that highly sulfated polysaccharides showed potent affinity irrespective of their polysaccharide structure. The sulfated chondroitin 4-sulfate having a sulfate content and molecular size comparable to those of heparin was inferior to heparin with respect to affinity. A competitive dissociation experiment indicated that heparin and other polysulfated polysaccharides share a common binding site on the fibronectin molecule.

Reactivity toward chemical sulfation of hydroxyl groups of heparin

Abstract The pyridinium salt of hog-mucosal heparin was desulfated by heating in 3:6:1 (v/v) 1,4-dioxane-dimethyl sulfoxide-methanol at 90° for 72 h, with the intermittent addition of pyridinium chloride (5 mol/mol of disaccharide). After N-resulfation, the desulfated polysaccharide (tributylammonium salt) was treated with 5–20 mol of pyridine-sulfur trioxide in N,N-dimethylformamide per mol equiv. of hydroxyl group at −10, 0, or 20° for 1 h to undergo O-sulfation. The sulfated products were quantitatively analyzed for 6-sulfate and 2-sulfate esters in 2-amino-2-deoxy- d -glucose and l -idosuronic acid units, respectively, by 13C- and 1H-n.m.r. spectroscopy and for total sulfate content by a chemical method. The new procedure gave 97% of desulfation of heparin, i.e., 100% N- and 6-desulfation of 2-amino-2-deoxy- d -glucose and 91.5% 2-desulfation of l -idosiduronic acid units. The O-resulfation proceeded according to the order of reactivity of the hydroxyl groups: HO-6 in GlcN ⪢ HO-2 in IdoA > other available hydroxyl groups (HO-3 in GlcN, HO-3 in IdoA, and HO-2 or HO-3 in GlcA).

Preferential formation of the hydroperoxide of linoleic acid in choline glycerophospholipids in human erythrocytes membrane during peroxidation with an azo initiator.

The formation of phospholipid hydroperoxides was monitored in human red blood cell (RBC) membranes that had been peroxidized with an azo initiator. Peroxidation of RBC membranes caused a profound decrease in the amount of polyunsaturated fatty acids and concomitantly hydroperoxides, as primary products of peroxidation, appeared in the phospholipids. Hydroperoxides were predominantly generated in choline glycerophospholipid (CGP), while the extent of formation of ethanolamine glycerophospholipid (EGP) hydroperoxides was low and their presence was transient. Hydroxy and hydroperoxy moieties in CGP were identified as 9-hydroxy and 13-hydroxy octadecanoic acid, derived from linoleic acid, by gas chromatography-mass spectrometric analysis. No consistent generation of hydroperoxide from arachidonic acid was evident in CGP. The CGP-hydroperoxide accounted for approximately 76% of linoleic acid consumed during peroxidation of RBC membranes. The prominent generation of phospholipid hydroperoxides was observed in the linoleic acid-rich membranes from rabbit RBC, indicating that the level of linoleic acid in phospholipids determines, in part, the extent of formation of phospholipid hydroperoxides. Aldehydic phospholipids, as secondary products of peroxidation, were detected in oxidized membranes. EGP was the most prominent aldehydic phospholipid, while negligible amounts of aldehydic CGP were formed. This study indicates that the process of oxidation of individual phospholipids clearly differs among phospholipids and depends on the structure of each.

Separation of heparin into fractions with different anticoagulant activity by hydrophobic interaction chromatography

Hog mucosal heparin purified on Sephadex G-100 (anticoagulant activity assayed by the method of the United States Pharmacopoeia, 179 units/mg) was separated by hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B into two groups, one with high affinity and another with low affinity for the gels. The former group was further separated into three fractions differing in hydrophobicity. The anticoagulant activities of the fractions with higher hydrophobicity ranged from 210 to 254 units/mg, whereas that of the fraction with lower hydrophobicity was 100 units/mg. The difference in antithrombin III-activation potency was much more prominent. The data obtained from affinity chromatography of these fractions on antithrombin III-Sepharose also substantiated the observed difference in anticoagulant activity. Analytical data of the fractions revealed a characteristic difference in both N-acetyl content and molecular size. While the N-acetyl content (mol/mol of hexosamine) and Kav value (on Ultrogel AcA44) of the fraction with the lowest hydrophobicity were 0.12 mol and 0.48, those of the fractions with higher hydrophobicity were 0.15-0.17 mol and 0.35-0.23, respectively.

Gas chromatographic determination of micro-amounts of cyclamates

It was found that triethylammonium cyclamate is converted into N-hepta-fluorobutyrylcyclohexylamine in a high and constant yield by reaction with hepta-fluorobutyric anhydride at 90 degrees for 1 h, and gas chromatography of the product gives a sharp peak that is highly sensitive to an electron capture detector. A useful method for the micro-determination of cyclamates was established by combining this reaction with gas chromatography.

Ion-exchange chromatography of nucleic acid constituents on chitosan-impregnated cellulose thin layers

Abstract A number of nucleic acid constituents, such as 5′-nucleotides, nucleosides and nucleic bases, could be separated and identified by chromatography on chitosan formate-impregnated cellulose thin layers. Suitable solvent systems and conditions for the chromatography were investigated, and RF data obtained for the compounds are given.

The structure of rooster-comb dermatan sulfate. Characterization and quantitative determination of copolymeric, isomeric tetra- and hexa-saccharides

Abstract Rooster-comb dermatan sulfates RC-20 and RC-30 were subjected to depolymerization-desulfation in hot dimethyl sulfoxide containing 10% of water to give almost quantitatively nonsulfated, even-numbered oligosaccharides having 2-acetamido-2-deoxy- d -galactose residues at the reducing terminals. They were fractionated on an anion-exchange resin with a linear gradient of lithium chloride into even-numbered oligosaccharide fractions from di- to dodeca-saccharide. Each fraction of di-, tetra-, and hexa-saccharide was isolated and further fractionated on an anion-exchange resin with a linear gradient of formic acid. All the oligosaccharide isomers that could theoretically exist (two disaccharide, four tetrasaccharide, and eight hexasaccharide fractions) were separated, and the sequences in disaccharide units were determined. The recoveries of the total amount of the di-, tetra-, and hexa-saccharide isomers isolated based on the starting RC-20 and RC-30 were 63.1 and 50.4%, respectively. The N -acetyldermosine-to- N -acetylchondrosine ratio in the disaccharide fractions from RC-20 and RC-30 had a value approximately equal to the IdoA-to-GlcA ratio. The yield of the hybrid oligomer containing N -acetylchondrosine and N -acetyldermosine was lower than expected, suggesting that the polysaccharide-chain structure of the dermatan sulfates (RC-20 and RC-30) of rooster comb contains many saccharide-chain blocks in which N -acetylchondrosine or N -acetyldermosine units are bonded sequentially.