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G David

Publications and source records attributed to G David.

At least 37 records · Page 2Linked to original sources
PubMed1995

Heparan sulfate proteoglycans. Essential co-factors in receptor-mediated processes with relevance to the biology of the vascular wall.

Heparan sulfate (HS), a mixed bag of complex, heterogeneous and highly charged polysaccharides, is an essential co-factor in a large number of receptor-ligand interactions and cellular pathways. These co-factor functions depend on the binding-interactions of the HS chains with the ligand or receptor, or both. These binding interactions and the ensuing functional effects often depend on defined carbohydrate sequences within the HS chains, whereby the required sequences are not always represented within all natural forms of the polysaccharide. The proteins that are substituted with HS resort from a limited number of protein families, with different cellular, subcellular and supramolecular associations, and show differential activities in functional assays. It is likely that the natural co-factor functions of the HS proteoglycans depend on glycan-protein and protein-protein interactions that are subject to modulation, both at the glycan and protein levels.

Animals↗
PubMed1994

Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis.

A survey of defined species of cell surface and extracellular matrix heparan sulfate proteoglycans (HSPG) was performed in a search for cellular proteoglycans that can promote bFGF receptor binding and biological activity. Of the various affinity-purified HSPGs tested, perlecan, the large basement membrane HSPG, is found to induce high affinity binding of bFGF both to cells deficient in HS and to soluble FGF receptors. Heparin-dependent mitogenic activity of bFGF is strongly augmented by perlecan. Monoclonal antibodies to perlecan extract the receptor binding promoting activity from active HSPG preparations. In a rabbit ear model for in vivo angiogenesis, perlecan is a potent inducer of bFGF-mediated neovascularization. These results identify perlecan as a major candidate for a bFGF low affinity, accessory receptor and an angiogenic modulator.

Animals↗
PubMed1994

Sulphated and undersulphated heparan sulphate proteoglycans in a Chinese hamster ovary cell mutant defective in N-sulphotransferase.

The Chinese hamster ovary cell mutant, pgsE-606, synthesizes undersulphated heparan sulphate glycosaminoglycans because of a deficiency in N-sulphotransferase activity [Bame and Esko (1989) J. Biol. Chem. 264, 8059-8065]. We compared the heparan sulphate proteoglycans synthesized by mutant and wild-type cells to determine what effect the undersulphation defect had on proteoglycan structure. The majority of heparan sulphate proteoglycans synthesized by pgsE-606 were undersulphated, but the mutant also synthesized a population of proteoglycans that were sulphated to the same extent as wild-type molecules. Anion-exchange analysis of the glycosaminoglycans in each proteoglycan population showed that they were all modified in the same way. The length of the glycosaminoglycans in each proteoglycan population were similar, suggesting that N-sulphation does not affect chain polymerization. To examine whether the sulphation state of the attached heparan sulphate glycosaminoglycans was dependent on the protein core, we purified syndecan-1 from mutant and wild-type cells using antibodies against the core protein. As with the unfractionated heparan sulphate proteoglycans, pgsE-606 synthesized both undersulphated and sulphated syndecan-1. Each pool contained either undersulphated or sulphated glycosaminoglycan chains respectively. Thus the modification of all heparan sulphate chains on a core protein occurs on a proteoglycan-wide basis (i.e. to the same extent).

Animals↗
PubMed1994

On the regional distribution of heparan sulfate proteoglycan immunoreactivity in the rat brain.

By means of two monoclonal antibodies specific for heparan sulfate (HS)-related epitopes, one (10E4) against native HS chains and one (3G10) against desaturated uronates, a highly regional and differential distribution of these two epitopes have been observed in the adult rat brain. The 10E4 epitope immunoreactivity (IR) is mainly found in the substantia nigra, the red nucleus and the subgranular zone of the dentate gyrus, while the 3G10 epitope IR is mainly found in the CA2 area of the hippocampal formation and the pyramdial cells in the layer V of the frontoparietal cortex. The codistribution of both types of IRs with basic fibroblast growth factor (bFGF, FGF-2) in neurons and astroglia supports the notion that heparan sulfate proteoglycans (HSPG) in the extracellular matrix may serve as a site for storage of bFGF and assist in the bFGF-induced activation of the high-affinity FGF receptors linked to astroglia and neurons in these discrete areas.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1994

Differential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor.

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). The capacity of various species of heparin and heparan sulfate (HS) to promote bFGF receptor binding was investigated using both Chinese hamster ovary mutant cells deficient in cell surface HSPG and a soluble bFGF receptor-alkaline phosphatase fusion protein. Highly sulfated oligosaccharides were more effective than medium and low sulfate fractions of the same size oligosaccharide. O-Sulfation in heparin was found to be critical for its capacity to promote binding of bFGF to its receptors. The highest level of bFGF-receptor binding was achieved in the presence of over-sulfated heparin fragments (% sulfur > 14) regardless of whether the N-position was sulfated or acetylated. Unlike receptor binding of bFGF which requires oligosaccharides containing at least 8-10 sugar units, displacement of heparin- or HS-bound bFGF was obtained by oligosaccharides containing as little as four sugar units and by an N-sulfated, O-desulfated heparin fragment (% sulfur = 5.3). A preparation of total cell surface-derived HS induced bFGF receptor binding. A preliminary survey of several defined and affinity purified species of cell surface HSPG, including syndecan, fibroglycan, and glypican failed to identify natural HSPG that promote high affinity receptor binding of bFGF. A similar lack of activity was observed with species of HS isolated from bovine arterial tissue and characterized for their effect on vascular smooth muscle cell proliferation. Moreover, most of these species of HS inhibited in a dose-dependent manner the restoration of bFGF-receptor binding induced by heparin or by total HSPG. These results suggest the involvement of defined heparin-like oligosaccharide sequences and unique species of cell surface and extracellular matrix HS in the regulation of bFGF receptor binding and biological activity.

Animals↗
PubMed1994

Non-covalent antibody-mediated drug delivery.

High background activity produces imaging problems when scanning with antibodies. The following work is directed towards reducing this background. The murine monoclonal antibody (MAb) CHA-255 selectively binds 111In-nitrobenzyl EDTA, a molecule referred to as a 'hapten'. Balb/c mice studies indicate that if the antibody is administered prior to the hapten, it predictably modifies the biodistribution and pharmacokinetics of the hapten. The pharmacokinetics for the hapten were proportional to antibody dose and inversely proportional to the time interval between injection of the antibody and the hapten. A hybrid MAb was produced by the enzymatic digestion of CHA-255 and ZCE-025, an anticarcinoembryonic antigen (CEA) MAb, followed by joining of the two via a thioether linkage. The result was a F(ab')2 with affinity for both CEA and the hapten. The pharmacokinetics of the hapten were again dependent upon the kinetics and distribution of the hybrid antibody. Data in tumour models are also presented for 111In-nitrobenzyl EDTA and 111In-thioureabenzyl EDTA (TUBE), a newer hapten. The data indicate that the antibody-hapten system is capable of targeting tumour quickly while normal tissue rapidly becomes depleted of radioactivity. We conclude that the hapten-antibody technique shows some advantages over directly labelled MAb as a targeting system.

Animals↗
PubMed1994

Differential expression of multiple cell-surface heparan sulfate proteoglycans during embryonic tooth development.

Heparan sulfate accumulates on cell surfaces and at cell-matrix interfaces, and functionally modulates several of the effector molecules that support the interactions, growth, and differentiation of developing tissues. Using heparin sulfate-specific monoclonal antibodies MAb, we obtained evidence that extracts from rodent embryos contain multiple forms of cell surface-associated heparan sulfate proteoglycan (PG). Taking tooth development in the mouse embryo as a model to further investigate the relevance of this PG redundancy and using MAb against heparan sulfate, antibodies specific for syndecan (syndecan-1) and fibroglycan (syndecan-2) (two distinct members of a larger family of cell-surface heparan sulfate PGs), and specific cDNA probes for these two cell-surface PGs, we obtained in situ evidence for regulated and differential expression of multiple cell-surface heparan sulfate PGs. The unique, distinctive, and coordinated changes in the expressions of these PGs during morphogenesis and differentiation of dental tissues suggest that the various cell-surface PGs are not truly redundant but play important, specific, and potentially complementary roles during embryonic development.

Animals↗
PubMed1994

Basic fibroblast growth factor-binding domain of heparan sulfate in the human glomerulosclerosis and renal tubulointerstitial fibrosis.

BACKGROUND: The saccharide side chains of heparan sulfate (HS) proteoglycans show enormous complexity. These polysaccharides can interact specifically with cytokines such as basic fibroblast growth factor (bFGF). The understanding of HS expression in glomerulosclerosis and interstitial fibrosis, which is still rudimentary, could provide some insight about the role of bFGF in kidney diseases. EXPERIMENTAL DESIGN: Kidney sections were exposed to exogenous bFGF and then to a monoclonal anti-bFGF antibody. Specificity of the interaction between HS and bFGF was established by monitoring concomitant loss of bFGF during selective removal of HS with heparitinase and competitive inhibition studies. To further characterize regional changes in saccharide sequences, heparitinase-generated unsaturated disaccharides, N-sulfated glucosamine-enriched but O-sulfate-scarce portions characteristics of native HS, and such portions characteristic of Engelbreth-Holm-Swarm tumor HS were studied. RESULTS: HS was detected in interstitial fibrosis and in advanced glomerulosclerosis, whereas bFGF-binding domains were found only in the fibrosis: The distributional pattern of the N-sulfate-enriched and O-sulfate-scarce portions of native HS was similar to that of bFGF-binding domains. Moreover, a small population of parenchymal cells in advanced tubulointerstitial fibrosis with marked cellular infiltration were especially rich in the bFGF-binding domains. CONCLUSIONS: In fibrotic lesions of the peritubular interstitium, HS shows enrichment of bFGF-binding domains. These regions may play an important role in the fibrogenesis through their interaction with endogenous bFGF.

Adult↗
PubMed1993

Integral membrane heparan sulfate proteoglycans.

Heparan sulfate is a regulatory polysaccharide. It modulates specific growth factor-receptor interactions, accelerates the formation of specific proteinase-proteinase inhibitor complexes, and mediates interactions of the cell surface with several enzymes and structural proteins. It abounds on the surfaces of embryonic cells, respecting or outlining morphogenetic rather than histological boundaries. This cell surface-associated heparan sulfate is implanted on specific integral membrane proteins, which together constitute two novel molecular families. The first family includes four syndecan-like integral membrane proteoglycans (SLIPS), with core proteins that span the membrane and shared sequence motifs in highly conserved cytoplasmic domains. The second is made up by two or more glypican-related integral membrane proteoglycans (GRIPS) that are linked to the cell surface via glycosyl phosphatidylinositol. These proteoglycans show differential expression and turnover patterns, prevailing in distinct cell types, membrane domains, and endocytotic machineries, and are subject to strict developmental controls. This suggests that each of these cell surface proteoglycans functions in a specific context, and that these functions pertain to the transduction of signals that emanate from the continuous interplay between matrix components, growth factors, and proteinases. Caution: beware of loose GRIPS and SLIPS on unsteady cell surfaces.

Amino Acid Sequence↗
PubMed1993

Activation of internodal potassium conductance in rat myelinated axons.

1. Voltage changes associated with currents crossing the internodal axolemma were monitored using a microelectrode inserted into the myelin sheath (peri-internodal region) of rat phrenic nerve fibres. This microelectrode was also used to change the potential and the ionic environment in the peri-internodal region. 2. Following stimulation of the proximal nerve trunk, the peri-internodal electrode recorded a positive-going action potential whose amplitude increased (up to 75 mV) with increasing depth of microelectrode penetration into the myelin. The resting potential recorded by the peri-internodal electrode remained within 4 mV of bath ground. 3. Confocal imaging of fibres injected peri-internodally with the fluorescent dye Lucifer Yellow revealed a staining pattern consistent with spread of dye throughout the myelin sheath of the injected internode. 4. After ionophoresis of K+ (but not Na+) into the peri-internodal region, the action potential was followed by a prolonged negative potential (PNP) lasting hundreds of milliseconds to several seconds. The duration of the PNP increased as the frequency of stimulation decreased. PNPs could also be evoked by sub-threshold depolarization of the internodal axolemma with peri-internodally applied current pulses. In the absence of action potentials or applied depolarization PNPs sometimes appeared spontaneously. 5. Peri-internodal application of Rb+ also produced evoked and spontaneous PNPs. These PNPs had longer durations (up to 20 s) than those recorded from K(+)-loaded internodes. 6. Spontaneous action potentials sometimes appeared during the onset of the PNP, suggesting that PNPs are associated with depolarization of the underlying axon. 7. Passage of current pulses during the PNP demonstrated that the PNP is associated with an increased conductance of the pathway linking the peri-internodal recording site to the bath. At least part of this conductance increase occurs across the internodal axolemma, since peri-internodally recorded action potentials evoked during the PNP had larger amplitudes than those evoked before or after the PNP. 8. PNPs were suppressed by tetraethylammonium (TEA, 10-20 mM) and by 4-aminopyridine (1 mM). 9. These results suggest that the PNPs recorded in K(+)- or Rb(+)-loaded myelin sheaths are produced by a regenerative K+ or Rb+ current that enters the internodal axolemma via K+ channels opened by action potentials or subthreshold depolarizations. 10. When normal extracellular [K+] was preserved (by using Na+ rather than K+ salts in the peri-internodal electrode), action potentials recorded within the myelin sheath were instead followed by a brief, positive after-potential that was inhibited by TEA.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1993

Posttetanic hyperpolarization produced by electrogenic Na(+)-K+ pump in lizard axons impaled near their motor terminals.

1. The hyperpolarization that follows tetanic stimulation was recorded intra-axonally from the internodal region of intramuscular myelinated motor axons. 2. The peak amplitude of the posttetanic hyperpolarization (PTH) that followed stimulation at 20-100 Hz for < or = 35 s increased with increasing train duration, reaching a maximum of 22 mV. PTH decayed over a time course that increased from tens to hundreds of seconds with increasing train duration. For a given frequency of stimulation the time integral of PTH was proportional to the number of stimuli in the train, averaging 3-4 mV.s per action potential. 3. Ouabain (0.1-1 mM) and cyanide (1 mM) depolarized the resting potential and abolished PTH. Tetanic stimulation in ouabain was followed by a slowly decaying depolarization (probably due to extra-axonal K+ accumulation) whose magnitude and duration increased as the duration of the train increased. 4. Axonal input resistance showed no consistent change during PTH in normal solution but increased during PTH in the presence of 3 mM Cs+ (which blocks axonal inward rectifier currents). 5. PTH was abolished when bath Na+ was replaced by Li+ or choline. PTH persisted after removal of bath Ca2+ and addition of 2 mM Mn2+. 6. Removal of bath K+ abolished the PTH recorded after brief stimulus trains and greatly reduced the duration of PTH recorded after longer stimulus trains. 7. A brief application of 10 mM K+, which normally depolarizes axons, produced a ouabain-sensitive hyperpolarization in axons bathed in K(+)-free solution. 8. These observations suggest that in these myelinated axons PTH is produced mainly by activation of an electrogenic Na(+)-K(+)-ATPase, rather than by changes in K+ permeability or transmembrane [K+] gradients. This conclusion is supported by calculations showing agreement between estimates of Na+ efflux/impulse based on PTH measurements and estimates of Na+ influx/impulse based on nodal voltage-clamp measurements. Pump activity also appears to contribute to the resting potential. 9. The stimulus intensity required to initiate a propagating action potential increased during PTH but decreased during the posttetanic depolarization recorded in ouabain. Thus changes in axonal excitability after tetanic stimulation correlate with changes in the posttetanic membrane potential. 10. Action potentials that propagated during PTH had a larger peak amplitude and were followed by a larger and longer depolarizing afterpotential than action potentials elicited at the resting potential. This enhancement of the depolarizing afterpotential is consistent with previous reports of an increased superexcitable period after action potentials evoked during PTH.

Animals↗
PubMed1993

Spatial and temporal changes in the expression of fibroglycan (syndecan-2) during mouse embryonic development.

Fibroglycan (syndecan-2) is a member of a family of cell surface heparan sulfate proteoglycans that interact with adhesion molecules, growth factors and a variety of other effector systems that support the shaping, maintenance and repair of an organism. To investigate this apparent redundancy of proteoglycans at the cell surface, we have studied the expression of fibroglycan in the mouse embryo and compared this expression with that of syndecan-1. The characterisation of mouse embryo cDNA clones that crosshybridized to human fibroglycan-cDNA predicted that murine and human fibroglycan were highly similar in structure. Consistently, the analysis of transfectant cells, murine cell lines and embryo extracts indicated that the murine proteoglycan reacted specifically with monoclonal antibody 10H4 developed against the human protein. Fibroglycan, as detected by monoclonal antibody 10H4 in sections of embryonic tissues, occurred exclusively on mesenchymal cells that represented the putative precursors of the hard and connective tissue cells. No fibroglycan was detected in epithelia or in muscle cells. Areas where fibroglycan was particularly abundant were sites of high morphogenetic activity where intense cell-cell and cell-matrix interactions are known to occur (e.g. the epithelial-mesenchymal interfaces, the prechondrogenic and preosteogenic mesenchymal condensations). The expression of fibroglycan was weak in the early embryo, culminated during the morphogenetic phase and at the moment of cell lineage differentiation, and persisted in the perichondrium, periosteum and connective tissue cells. Syndecan-1, in contrast, was primarily detected in epithelia, and transiently in some mesenchymal cells, with mesenchymal localisations that did not or only partially overlap with those of fibroglycan. In situ hybridization analyses confirmed these expression patterns at the transcriptional level, identifying mesenchymal cells as the major source of fibroglycan production. These data indicate that the expression of fibroglycan occurs along unique and developmentally regulated patterns, and suggest that fibroglycan and syndecan-1 may have distinctive functions during tissue morphogenesis and differentiation.

Amino Acid Sequence↗
PubMed1993

[A new entity of sperm pathology: peri-axonemal flagellar dyskinesia].

The study of 17 infertile men has led to define a new entity of sperm pathology as part of the more general field of flagellar dyskinesias. Sperm parameters of the studied patients and a control series have been first estimated by routine analysis (concentration, motility, morphology). To precise their characteristics, kinetic and ultrastructural investigations, as the zona-free hamster oocyte penetration test, have been performed. Sperm parameters of the studied cases, as revealed by routine analysis, were close to the control group. However, a major kinetic anomaly was found which was characterized by an important decrease of the amplitude of lateral head displacement (1.6 microns vs 5.3 microns, p < 0.001), although the progressive velocity was only slightly impaired (20.3 microns vs 24.9 microns, p < 0.05). Electron microscopy revealed anomalies limited to the peri-axonemal structures such as the outer dense fibers and the fibrous sheath. Rates of sperm-oocyte attachment were normal but rates of oocyte penetration were low (27.7% of decondensed sperm heads vs 85.6%, p < 0.001). Attempts to assisted fertilization with the studied patients (51 cycles of insemination, 8 cycles of in vitro fertilization) were unsuccessful. All these data suggest that the infertility can be attributed to the movement disturbances which should impair sperm propulsion throughout the cervical mucus and the zona pellucida.

Animals↗
PubMed1992

Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties.

Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall.

Antithrombin III↗
PubMed1992

Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts.

Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.

Amino Acid Sequence↗