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R J Linhardt

Publications and source records attributed to R J Linhardt.

At least 181 records · Page 10Linked to original sources
PubMed1986

Effect of very low molecular weight heparin-derived oligosaccharides on lipoprotein lipase release in rabbits.

Oligosaccharide fragments of heparin were prepared using flavobacterial heparinase. Following sizing, these oligosaccharide fractions were administered (i.v.) to rabbits and were examined for their ability to release lipoprotein lipase. The decasaccharides (dp = 10, Mr avg = 2,800) were the smallest oligosaccharides which resulted in substantial lipase release. The plasma lipase levels obtained with decasaccharides were comparable to low molecular weight heparin and one-third those obtained when heparin was administered at an equivalent dose. The peak plasma lipase concentration was observed 10 min following heparinization and fell off rapidly over the 60-min time course. The lipase release activity paralleled the in vivo pharmacokinetics of the heparin and decasaccharide sample as determined by monitoring their anti-Factor Xa activity. No activation of purified bovine milk lipoprotein lipase or plasma lipase was detectable at the concentrations studied, indicating that the increase in circulating lipolytic activity was due entirely to release. Lipoprotein lipase accounted for a major portion of the released activity with hepatic triglyceride lipase representing the remainder of the lipolytic activity. The sized decasaccharide sample was characterized with regards to its structure and anticoagulant activity. The decasaccharides exhibited reduced anticoagulant activity possibly making it a better drug candidate in the treatment of atherosclerosis.

Animals↗
PubMed1986

Effect of heparin, heparin fragments, and corticosteroids on cerebral endothelial cell growth in vitro and in vivo.

Heparin and heparin fragments in combination with corticosteroids have been shown to markedly inhibit tumor angiogenesis. Experiments were performed to test the hypothesis that heparin, heparin fragments, and the combination of heparin and corticosteroids affect DNA synthesis and the proliferation of cerebral microvessel endothelium (ME). In vitro, methyl-3H-thymidine incorporation in the ME cells was measured after a 24 hour pulse. Our results show that heparin, hydrocortisone, and heparin in combination with hydrocortisone had a slight inhibitory effect on DNA synthesis of ME (p less than 0.05), and hydrocortisone in combination with heparin had a slight inhibitory effect on ME cell growth (p less than 0.05). The hexa-, octa-, and deca-saccharide fragments of heparin stimulated DNA synthesis in ME (p less than 0.01). In vivo, DNA synthesis in cerebral endothelial cells at the margin of a freeze lesion to mouse cerebral cortex was assayed by quantitation of labeling indexes from methyl-3H-thymidine autoradiographs in mice treated with heparin, cortisone, or a combination of heparin and cortisone. A mean endothelial cell labeling index (LI) of 6% in the cortisone-treated animals was significantly lower than controls (32%, p less than 0.01). The addition of heparin to cortisone did not significantly alter the endothelial cell LI compared to the cortisone-treated animals, and heparin alone did not significantly alter the LI compared to the controls. These results indicate that cortisone markedly reduces the endothelial proliferation around a cortical freeze lesion in vivo. This effect is independent of heparin.

Adrenal Cortex Hormones↗
PubMed1986

Variation in composition and yield of exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4.

The exopolysaccharides produced by Klebsiella sp. strain K32 and Acinetobacter calcoaceticus BD4 under different growth conditions have been analyzed for sugar composition. The first use of ion chromatography for the quantitative determination of microbial exopolysaccharide composition is reported. Klebsiella sp. strain K32 produced a polymer composed of rhamnose, galactose, and mannose early in its fermentation. The composition of the polymer varied markedly depending on the growth stage of the organism. Klebsiella sp. strain K32 grown in a fermentor produced a polymer which was rich in mannose during early exponential growth in a complex medium, but in the late stationary phase it did not contain detectable levels of mannose. The rhamnose present in the polymer increased from 12 to 55% over the course of growth, whereas galactose decreased from 63 to 45%. A. calcoaceticus BD4 produced a polymer containing rhamnose, glucose, mannose throughout its growth and stationary phase. Klebsiella sp. strain K32 and A. calcoaceticus BD4 were grown on various carbon sources in shake flasks. The polymer yield and composition from both organisms were found to vary with the carbon source. The exopolysaccharide with the highest mannose composition was obtained by using rhamnose as a carbon source for both organisms. These and other data suggest that regulatory changes caused by growth on different substrates result in either the production of a different distribution of polymers or a change in exopolysaccharide structure.

Acinetobacter↗
PubMed1985

Evidence of random structural features in the heparin polymer.

The first use of computer-simulation studies to examine heparin's structure has been reported. The product distributions obtained when porcine mucosal heparins were depolymerized with heparinase have been compared to computer-simulated distributions. The modeled distribution was relatively unaffected by the polydispersity and molecular weight of heparin. However, the percent of heparinase-cleavable glycosidic linkages and their distribution throughout the polymer resulted in a marked change in the simulated product distribution. The similarity between experimentally observed and computer-simulated product distributions is consistent with the random distribution of heparinase-cleavable sites in porcine mucosal heparin. Finally, a random distribution of N-acetyl residues with respect to heparinase-cleavable sites was experimentally observed.

Animals↗
PubMed1985

High-performance liquid chromatographic separation of heparin-derived oligosaccharides.

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gel-permeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.

Chromatography, Gel↗
PubMed1985

Structure of heparin-derived tetrasaccharides.

The structure of heparin was examined by characterizing a disaccharide and five of the more than a dozen tetrasaccharide components obtained by its depolymerization with flavobacterial heparinase. Enzymic depolymerization of porcine mucosal heparin results in a mixture of di-, tetra-, hexa- and higher oligo-saccharides. The di- and tetra-saccharide components represent 75mol/100mol of these heparin fragments. Ion-exchange chromatography indicates the presence of only one disaccharide, deltaIdu2S(1----4)-alpha-D-GlcNS6S (where Idu is iduronic acid, deltaIdu is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is glucosamine, GlcA is glucuronic acid and S is sulphate), but results in the isolation of five major and at least seven minor tetrasaccharide components. The structures of the disaccharide and five major tetrasaccharides were determined by chemical, enzymic, electrophoretic and spectroscopic methods, including 13C, 1H n.m.r. and fast atom bombardment-m.s. The structure of these five tetrasaccharides are: delta Idu2S(1----4)-alpha-D-GlcNS6S(1---4)-alpha-L-Idu2S(1-- --4)-alpha -D-GlcNS6S; delta Idu2S(1----4)-alpha-D-GlcNS6S(1----4)-beta-D-GlcA(1--- -4)- alpha-D-GlcNS6S; delta Idu2S(1----4)-alpha-D-GlcNS(1----4)-beta-D-GlcA delta Idu2S(1----4)-alpha-D-GlcNAc(1----4)-beta-D-GlcA(1----4)- alpha-D-GlcNS6S; and delta Idu2S(1----4)-alpha- D-GlcNAc(1----4)-alpha-L-Idu(1----4)-alpha-D-GlcNS6S. Biological activity for the disaccharide and the five major tetrasaccharides was examined, and none of them were found to possess significant anticoagulant activity.

Animals↗
PubMed1985

Purification and characterization of heparinase from Flavobacterium heparinum.

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.

Amino Acids↗
PubMed1985

Immuno-affinity purification of heparinase.

Polyclonal IgG rabbit antibodies were prepared against a purified heparinase from Flavobacterium heparinum. Immuno-affinity purification of crude and partially purified heparinase is described. The resulting enzyme was of comparable purity to that prepared using the standard multistep purification scheme. The antibodies prepared were found to increase the activity of bound heparinase.

Animals↗
PubMed1985

Small heparin fragments regulate the amplification pathway of complement.

Heparin is a highly sulfated, polydisperse and heterogeneous glycosaminoglycan which has been well characterized for its ability to regulate multiple sites in the complement cascade. Although previous studies demonstrated the relationship between degree of sulfation, particularly O-sulfation, and complement inhibiting capacity, they left unclear the relationship between the size of the heparin molecule and its ability to inhibit complement. Therefore, although the structure-activity relationship for heparin is well understood for anticoagulant activity, it is ill defined for the complement system. The present studies were designed to examine depolymerized heparin to determine which fragments were capable of inhibiting amplification pathway activation. We found that as the size of the molecule increases the ability to regulate complement increases; below 1000 Da the fragments were essentially inactive and above 3500 Da they had the same activity as does commercial heparin. Furthermore, we examined the five major tetrasaccharides of heparin and found that the degree of sulfation did correlate with the ability to inhibit complement. These studies have for the first time begun to examine the minimal structural requirements for heparin to regulate complement.

Complement Activation↗
PubMed1984

An immobilized microbial heparinase for blood deheparinization.

A new medical application of an immobilized microbial enzyme is described. Extracorporeal devices require systemic heparin administration to prevent thrombus formation; however, the use of heparin often leads to serious hemorrhagic complications. Heparinase isolated from Flavobacterium has been immobilized and used in a fluidized bed reactor to eliminate heparin from blood passing through an extracorporeal circuit both in vitro and in vivo. This paper discusses the stepwise development of this heparinase reactor including: (1) improvements in the fermentation resulting in an inexpensive large-scale source of heparinase without the addition of the previously required inducer, heparin; (2) the use of batch processes to adapt previous purification schemes to large-scale heparinase production and the subsequent purification of heparinase to a single SDS-PAGE banding protein; (3) the immobilization of heparinase with a 91% activity recovery and good stability, (4) the design and successful testing of a fluidized bed reactor containing immobilized heparinase in the removal of clinically used quantities of heparin from both human blood in vitro and canine blood in vivo; and (5) the initiation of animal studies focusing on the toxicology of heparinase-derived heparin degradation products and the short and long term effects of exposure to these products and to heparinase.

Enzymes, Immobilized↗