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D J Tyrrell

Publications and source records attributed to D J Tyrrell.

13 recordsLinked to original sources
PubMed1999

Heparin in inflammation: potential therapeutic applications beyond anticoagulation.

In this chapter we have described anti-inflammatory functions of heparin distinct from its traditional anticoagulant activity. We have presented in vivo data showing heparin's beneficial effects in various preclinical models of inflammatory disease as well as discussed some clinical studies showing that the anti-inflammatory activities of heparin may translate into therapeutic uses. In vivo models that use low-anticoagulant heparins indicate that the anticoagulant activity can be distinguished from heparin's anti-inflammatory properties. In certain cases such as hypovolemic shock, the efficacy of a low-anticoagulant heparin derivative (GM1892) exceeds heparin. Data also suggest that nonconventional delivery of heparin, specifically via inhalation, has therapeutic potential in improving drug pharmacokinetics (as determined by measuring blood coagulation parameters) and in reducing the persistent concerns of systemic hemorrhagic complications. Results from larger clinical trials with heparin and LMW heparins are eagerly anticipated and will allow us to assess our predictions on the effectiveness of this drug class to treat a variety of human inflammatory diseases.

Alzheimer Disease↗
PubMed1996

Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties.

Structural features of heparin potentially important for heparanase-inhibitory activity were examined by measuring the ability of heparin derivatives to affect the degradation of [3H]acetylated heparan sulphate by tumor cell heparanases. IC50 values were determined using an assay which distinguished degraded from undegraded substrate by precipitation of the latter with cetylpyridinium chloride (CPC). Removal of heparin's 2-O-sulphate and 3-O-sulphate groups enhanced heparanase-inhibitory activity (50%). Removal of its carboxyl groups slightly lowered the activity (18%), while combining the treatments abolished the activity. At least one negative charge on the iduronic acid/idose moiety, therefore, is necessary for heparanase-inhibitory activity. Replacing heparin's N-sulphate groups with N-acetyl groups reduced its activity (37%). Comparing this heparin derivative with 2,3-O-desulphated heparin, the placement of sulphate groups appears important for activity since the two structures have similar nominal linear charge density. In addition, unsubstituted uronic acids are nonessential for inhibition since their modification (periodate-oxidation/borohydride-reduction) enhanced rather than reduced heparanase-inhibitory activity. The most effective heparanase inhibitors (2,3-O-desulphated heparin, and [periodate-oxidized, borohydride-reduced] heparin) were tested in the chick chorioallantoic membrane (CAM) bioassay for anti-angiogenic activity and found to be at least as efficacious as heparin. 2,3-O-desulphated heparin also significantly decreased the tumor growth of a subcutaneous human pancreatic (Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival times of C57BL/6N mice in a B16-F10 melanoma experimental lung metastasis assay.

Animals↗
PubMed1993

Preparation of affinity-fractionated, heparin-derived oligosaccharides and their effects on selected biological activities mediated by basic fibroblast growth factor.

Homogeneously sized, heparin-derived oligosaccharides were prepared from heparin following partial depolymerization with nitrous acid, reduction with sodium borohydride, and fractionation by gel permeation chromatography. The resulting pools of di-, tetra-, hexa-, octa-, and decasaccharides were sequentially applied to an affinity column of human recombinant basic fibroblast growth factor (bFGF) covalently attached to Sepharose 4B and further fractionated into subpools based on their elution from this column in response to gradients of sodium chloride. In general, pools of smaller heparin-derived oligosaccharides required relatively lower salt concentration for complete elution, and pools of larger oligosaccharides required higher salt concentration. The homogeneously sized pools and affinity-fractionated subpools of heparin-derived oligosaccharides were quantitatively assessed as inhibitors or enhancers of specific bFGF-mediated biological activities in five separate assay systems as follows: assay 1, to compete with human lymphoblastoid cells expressing syndecan (RO-12 UC cells) for binding to bFGF-coated wells (Ishihara, M., Tyrrell, D.J., Kiefer, M.C., Barr, P.J., and Swiedler, S.J. (1992) Anal. Biochem. 202, 310-315); assay 2, to inhibit 125I-bFGF binding to "low affinity sites" of adrenocortical endothelial (ACE) cells; assay 3, to inhibit bFGF-induced proliferation of ACE cells; assay 4, to support mitogenic activity of bFGF in a growth stimulation assay of chlorate-treated ACE cells; and assay 5, to enhance the in vitro interaction between 125I-bFGF and the recombinant extra-cellular domain of FGF high affinity receptor. The data derived from the five assay systems demonstrated that heparin-derived hexa- and octasaccharides inhibited the interaction between cell surface heparan sulfate proteoglycan and bFGF (assays 1 and 2) and bFGF-induced proliferation of ACE cells (assay 3) but were unable to enhance the binding of bFGF to its high affinity receptor in vitro (assay 5) or to support bFGF-induced mitogenesis in ACE cells (assay 4). These two activities required at least a decasaccharide with high affinity for bFGF.

Animals↗
PubMed1993

Structure and biological activities of a heparin-derived hexasaccharide with high affinity for basic fibroblast growth factor.

We demonstrated previously that heparin-derived hexasaccharides are the smallest fragments of the polysaccharide with comparable basic fibroblast growth factor (bFGF)-modulating activity in vitro (Ishihara, M., Tyrrell, D.J., Stauber, G.B., Brown, S., Cousens, L., and Stack, R.J. (1993) J. Biol. Chem. 268, 4675-4683. In this report, a specific hexasaccharide having high affinity for recombinant human bFGF was isolated and its structure deduced by analysis of its reduced disaccharide products after treatment with nitrous acid at pH 1.5, and by 1H NMR spectroscopy. The hexasaccharide has the structure [IdoA(2-OSO3)alpha 1-4GlcNSO3(6-OSO3)alpha 1-4]2IdoA(2-OSO3)alpha 1-4 AManR(6-OSO3). The hexasaccharide effectively inhibits the binding of syndecan-transfected RO-12 UC cells to bFGF-coated wells (Ishihara, M., Tyrrell, D.J., Kiefer, M.C., Barr, P.J., and Swiedler, S.J. (1992) Anal. Biochem. 202, 310-315), prevents the binding of 125I-bFGF to confluent monolayers of adrenocortical endothelial (ACE) cells, and inhibits the bFGF-dependent proliferation of ACE cells. Unlike the heparin from which it was derived, however, the hexasaccharide cannot promote the binding of 125I-bFGF to a recombinant high affinity bFGF receptor (flg) or restore the bFGF-dependent proliferative response to ACE cells grown in the presence of 5 mM sodium chlorate. Collectively, these data indicate that a hexasaccharide can be as effective as heparin as an antagonist of bFGF-mediated cell mitogenesis.

Animals↗
PubMed1992

A cell-based assay for evaluating the interaction of heparin-like molecules and basic fibroblast growth factor.

A simple panning procedure that allows for the evaluation of interactions between various heparin-like molecules and basic FGF has been developed. This assay measures the ability of compounds to inhibit the interaction of transfected human lymphoblastoid cells, UC 729-6 (UC cells), expressing hamster syndecan and basic FGF-coated plastic plates. The transfected cells bind rapidly to basic FGF-coated plates while the control cells do not bind well. Binding of the transfected cells to basic FGF was inhibited by heparin and heparin sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. There was little inhibition of binding by chemically modified heparin such as completely desulfated, N-acetylated heparin, completely desulfated, N-sulfated heparin, and N-desulfated, N-acetylated heparin. These results suggested that both the N-sulfate and O-sulfate groups of heparin are required for binding to basic FGF. In addition, inhibition by oligosaccharides derived from depolymerized heparin increased with fragment size; partial inhibition was observed with oligosaccharides as small as hexamers. The biochemical basis for the binding of transfected cells to basic FGF was established by showing a significant increase of 35SO4 incorporation into HS. In particular, the level of 35SO4-HS in the trypsin-releasable (cell surface) pool increased fivefold. This increase was accounted for by demonstration of the presence of HS on immunoprecipitated syndecan from the transfected cells.

Animals↗
PubMed1988

Fibronectin is a component of the sodium dodecyl sulfate-insoluble transglutaminase substrate.

Liver plasma membranes contain a morphologically distinct protein complex which serves as a substrate for the plasma membrane-associated transglutaminase. The complex, which appears as a two-dimensional sheet, is insoluble in sodium dodecyl sulfate and reducing agents and has been named SITS for sodium dodecyl sulfate-insoluble transglutaminase substrate (Tyrrell, D. J., Sale, W. S., and Slife, C. W. (1988) J. Biol. Chem. 263, 1946-1951). Polyclonal antibodies raised against SITS were used to probe for soluble constituents of the matrix. Immunoblots showed that proteins of 230, 35, and 32 kDa reacted with the anti-SITS antiserum when the soluble fraction from a liver homogenate was examined. The 230-kDa protein was identified as fibronectin after observing cross-reactivity of anti-SITS antiserum with authentic fibronectin and cross-reactivity of anti-fibronectin antiserum with the 230-kDa cytosolic protein and purified SITS. Preincubating anti-SITS antiserum with purified fibronectin decreased immunostaining of the 230-kDa cytosolic protein and authentic fibronectin. Immunoblots of the plasma membrane fraction using anti-SITS and anti-fibronectin antisera showed that both antisera reacted with proteins at the top of the stacking gel (SITS) and of 230 kDa. In addition, the anti-SITS antiserum reacted with proteins of 85, 35, and 32 kDa. Immunofluorescence microscopy revealed that the anti-SITS and anti-fibronectin antisera both react with isolated SITS and with the same filamentous structures associated with intact plasma membranes. These studies show that fibronectin is a component of the plasma membrane matrix, SITS. This finding is consistent with the proposed role of this matrix which is to mediate cell-cell adhesion between hepatocytes in the tissue.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

Isolation of a sodium dodecyl sulfate-insoluble transglutaminase substrate from liver plasma membranes.

Rat liver plasma membranes contain transglutaminase activity and a large molecular weight protein complex which serves as a substrate for this enzyme. When plasma membranes were solubilized in sodium dodecyl sulfate and disulfide-reducing agents the transglutaminase substrate was recovered in the detergent-insoluble fraction. The insolubility of the complex suggested that it might be further studied by adsorbing membranes onto glass slides, then extracting with the detergent and reducing agent. After extraction, dark field light microscopy revealed numerous flattened sheets which varied in size from 4 to 12 micrometers. To confirm that these structures were the large molecular weight transglutaminase substrate, the plasma membranes were solubilized in sodium dodecyl sulfate and dithiothreitol and sedimented through a sucrose gradient containing the agent. The large molecular weight substrate was the only material found at the 1.11/1.23 g/cm3 interface. Microscopic examination showed the same structures previously observed on the glass slides. We conclude that the large molecular weight transglutaminase substrate is a sodium dodecyl sulfate-insoluble, morphologically distinct, protein complex. Due to its considerable size, nondissociable nature, and association with the lateral membrane, the sodium dodecyl sulfate-insoluble transglutaminase substrate may serve as a type of skeleton or scaffolding for this plasma membrane domain.

Animals↗
PubMed1986

Localization of a liver transglutaminase and a large molecular weight transglutaminase substrate to a distinct plasma membrane domain.

Rat liver plasma membranes contain transglutaminase activity and a large molecular weight protein aggregate that serves as a substrate for this enzyme (Slife, C.W., Dorsett, M.D., Bouquett, G.T., Register, A., Taylor, E., and Conroy, S. (1985) Arch. Biochem. Biophys. 241, 329-336; Slife, C.W., Dorsett, M.D., and Tillotson, M.L. (1986) J. Biol. Chem. 261, 3451-3456). When purified plasma membranes were sonicated and the different plasma membrane domains were separated by sedimentation through a linear sucrose gradient, virtually all of the transglutaminase activity and the large molecular weight transglutaminase substrate were associated with membrane fragments which migrated to a very dense region of the gradient (1.18 g/cm3). The bile canalicular markers, 5'-nucleotidase and HA-4 antigen, were predominantly found at 1.11 g/cm3, while most of the sinusoidal/lateral marker, CE-9 antigen, was detected at 1.14 g/cm3. Smooth membrane vesicles were observed chiefly at the lighter densities upon morphological analysis, while many filament-bearing, plasma membrane segments and junctional complexes were contained in the heavy transglutaminase fractions. These data show that the plasma membrane transglutaminase and the large molecular weight transglutaminase substrate are associated with a distinct region of the plasma membrane.

Animals↗
PubMed1986

Cytochrome P-450scc-substrate interactions. Role of the 3 beta- and side chain hydroxyls in binding to oxidized and reduced forms of the enzyme.

The steroid binding specificity of cytochrome P-450scc has been investigated for different oxidation/reduction and ligand-binding states of the enzyme (oxidized, reduced, oxygen-bound, and carbon monoxide-bound forms). The oxygen of the 3 beta-hydroxyl of cholesterol is important for the initial enzyme-substrate interaction. Significant binding requires the correct stereochemistry and appears to be controlled by the electron density on the 3 beta-oxygen. Interactions at this position (located at least 13 A from the heme iron) can modulate the heme midpoint potential. The binding site in this region contains a cleft which can accommodate up to two carbons joined in an ether linkage to the 3 beta-oxygen. The steroid intermediates of side chain cleavage (22R-hydroxycholesterol and 20 alpha,22R-dihydroxycholesterol) bind more tightly to the ferric enzyme than does cholesterol and utilize specific interactions of these side chain hydroxyls with a grouping(s) on the polypeptide chain (i.e. not with the heme iron). The interaction requires the correct stereochemistry; a 22S-hydroxyl cannot be readily accommodated in the binding site. The specificity of the interaction for hydroxyls at the 22R- versus the 20 alpha-position is altered upon reduction of the enzyme, indicating a reduction-induced conformational change in the active site. The specific interference of binding of 22R-hydroxy steroids by heme-bound carbon monoxide (but not oxygen), together with the known bond angles and distances for Fe-C-O and Fe-O-O, allows localization of the 22R-hydroxyl group on a line perpendicular to the heme plane, between 2 and 3 A from the iron.

Animals↗
PubMed1981

Input and distractor modality effects upon the release from proactive interference in children.

Build-up of proactive interference (PI) with visual-picture and auditory-verbal input modalities and the subsequent release from Pi following a change in modality was investigated in three experiments with boys and girls, as follows: Experiment I (n=64) at two mean age levels, 7-6 and 10-5; Experiment II (n=64) at mean age 7-6; and Experiment III (n=48) at age 11-4. PI build-up occurred in both modalities for all ages tested. Release from PI occurred following a change from auditory to visual input by not from PI occurred following a change from auditory shift. In the final experiment, this asymmetrical improvement in performance was dependent upon an interaction between the modality of the input and distractor task on the final or release trial; changing to visual input produced a release effect regardless of the distractor task modality, while auditory input was associated with improvement in recall if a visual distractor task was employed whether or not a shift in input modality had occurred. This improvement was hypothesized to represent a decrease in retroactive interference rather than a release from proactive interference.

Child↗
PubMedDate not supplied

The effect of inhaled heparin and related glycosaminoglycans on allergen-induced eosinophil infiltration in guinea-pigs.

Aerosolized unfractionated heparin GM1060 significantly inhibited allergen-induced eosinophil infiltration into the airways of guinea-pigs (as assessed both histologically and by bronchoalveolar lavage, or BAL) at doses of 160 and 1600 U/ml. Similarly aerosolized unfractionated heparin, Multiparin was effective at reducing eosinophil levels in the BAL fluid at 1000, 2000 and 5000 U/ml, but this reduction was statistically significant only at the highest dose used. Additionally, Fragmin (a low molecular weight heparin) significantly inhibited allergen-induced eosinophil infiltration into BAL fluid at a dose of 500 U/ml, an effect that was lost at the higher doses of 1000 and 2000 U/ml. Allergen-induced eosinophil infiltration was unaffected by dermatan sulphate. However, the glycosaminoglycans chondroitin sulphate A, chondroitin sulphate C and heparan sulphate were able to influence the extent of allergen-induced eosinophil infiltration into BAL fluid. These results suggest that heparin and some related glycosaminoglycans can inhibit allergen-induced eosinophil infiltration when administered directly to the airways.

Administration, Inhalation↗