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Biomedical subjects

J T Gallagher

Publications and source records attributed to J T Gallagher.

At least 55 records · Page 3Linked to original sources
PubMed1995

Receptors for fibroblast growth factors.

The recent discovery of the involvement of heparan sulfate proteoglycans (HSPG) in the activation of fibroblast growth factor receptors (FGFR) has led to an intensification of study of this field. It appears that the HSPG act as low affinity receptors to which the fibroblast growth factors (FGF) must bind in order to successfully activate the high affinity FGFR. Heparan sulfate chains consisting of alternately arranged N-acetylated or N-sulfated glucosamine and uronic acid disaccharide regions, covalently attached to a core protein are found in two major families of cell surface HSPG, the syndecans and glypicans. A high affinity bFGF binding region has been isolated from fibroblast HS. There are four basic members of the FGFR family (FGFR 1-4), as well as a wealth of splice variants. The alternative forms of the basic receptors can have altered ligand binding or signalling qualities, depending on the region of the gene which is spliced. Investigations with null FGFR, incapable of signalling, have demonstrated the requirement for FGF in the organization of mammalian tissues and in embryonic patterning. Mutation of the FGFR genes has been recognized recently in human craniosynostoses where a single base pair mutation in the FGFR gene results in skeletal malformations specific to each syndrome. One suggestion is that the interaction of the mutant FGFR with the HSPG/FGF complex somehow contributes to the disease phenotype.

Binding Sites↗
PubMed1995

A Badal optical stimulator for the Canon AutoRef R-1 optometer.

The Canon AutoRef R-1 optometer is currently in use by several investigators in studies of accommodative response and open loop accommodation. It has the capability for frequent (1.4 s cycle time in "single shot" mode) objective measures of the refractive state of the eye and, because it uses near infrared radiation, it provides no stimulus to accommodation. In this paper we describe a compact device which can be mounted on the R-1 optometer, coaxial with the measurement optics of the optometer, to provide controlled optical vergence, i.e., accommodative stimulus, from +10.0 to -20.0 D while maintaining a constant visual angle.

Accommodation, Ocular↗
PubMed1994

Alpha- and beta-xylosides modulate the synthesis of fibronectin and thrombospondin-1 by endothelial cells.

We have previously shown that both p-nitrophenyl-alpha-D-xylopyranoside (alpha-xyloside) and p-nitrophenyl-beta-D-xylopyranoside (beta-xyloside) inhibit endothelial morphogenesis in vitro. In order to determine the mechanism for this inhibition, we have now investigated the effects of these compounds on the synthesis of proteoglycans and proteins by bovine aortic endothelial cells. Consistent with their well-recognised modes of action, beta-xyloside, but not alpha-xyloside, enhanced the secretion of free glycosaminoglycans into the medium. Furthermore, although both xylosides inhibited proteoglycan deposition into the cell layer/matrix, only beta-xyloside altered the nature of the proteoglycans synthesised by the cells. Both alpha- and beta-xylosides markedly inhibited total protein synthesis by endothelial cells in the absence of any effect on cell growth. This inhibition was time- and dose-dependent and was not due to the enzymatic release of p-nitrophenol by the cells. The synthesis of fibronectin and thrombospondin-1 were specifically and differentially modulated by both alpha- and beta-xylosides. That is, xylosides markedly reduced fibronectin levels relative to other proteins in both the medium and the cell layer/matrix. In contrast, the relative levels of thrombospondin-1 were increased in the xyloside-treated cultures both in terms of mRNA and protein. These studies demonstrate novel effects of xylosides on protein synthesis. Furthermore, they suggest that the inhibition of endothelial morphogenesis by xylosides may be due to the actions of these compounds on the synthesis of specific proteins.

Animals↗
PubMed1994

Cell surface syndecan-1 on distinct cell types differs in fine structure and ligand binding of its heparan sulfate chains.

Heparan sulfate (HS) can bind a large variety of biological effectors, including extracellular matrix components, growth factors, chemokines, degradative enzymes, and protease inhibitors. Where studied, HS is known to be structurally heterogeneous and to vary in sulfation pattern between cells and tissues. Because heparan sulfate can represent several distinct proteoglycans, we asked whether the structural variation in the heparan sulfate chains of a single species of cell surface proteoglycan is a reproducible, differentiated characteristic and whether the variation can result in distinct biological functions. We studied the molecular structure and binding affinity for type I collagen and fibroblast growth factor-2 of syndecan-1 purified from the surfaces of NMuMG normal murine mammary gland epithelia, NIH/3T3 fibroblasts, and BALB/3T3 endothelioid cells. Syndecan-1 from these cell types varied in molecular mass largely due to variation in the length of the HS chains. Although the highly sulfated and N-acetylated domains in these HS chains were organized similarly, the number of highly sulfated domains differed. The disaccharide compositions were also similar except for reproducible and consistent differences in the amount of hexuronic acid-N-sulfated-6-O-sulfated glucosamine and 2-O-sulfated hexuronic acid-N-sulfated glucosamine. These differences were confirmed by oligosaccharide mapping, which showed cell type-specific variations in the composition of the highly sulfated domains. These structural variations correlated with cell type-specific differences in the affinity of syndecan-1 and its isolated HS chains for type I collagen. However, no differences in affinity for fibroblast growth factor-2 were detected. The results indicate that the size, fine structure, and ligand affinity of the HS chains on a single proteoglycan species differ in a consistent and reproducible manner between cell types. Thus, the variation in structure and binding ability of HS on syndecan-1 is a differentiated characteristic of the cell type that can enable cells to respond distinctly to the HS-binding effectors in the cellular microenvironment.

3T3 Cells↗
PubMed1994

Fine structure of heparan sulfate regulates syndecan-1 function and cell behavior.

Two myeloma cell lines, MPC-11 and P3X63Ag8.653 (P3), have almost identical amounts of syndecan-1 at their cell surface. The syndecan-1 molecules from both lines are similar in size, have indistinguishable core proteins, and have similarly sized heparan sulfate chains. Nevertheless, syndecan-1 on MPC-11 mediates cell adhesion to type I collagen, whereas P3 cells do not bind collagen. Affinity co-electrophoresis reveals that intact syndecan-1 isolated from P3 cells binds collagen poorly and that syndecan-1 heparan sulfate isolated from MPC-11 has a 20-fold higher affinity for collagen than syndecan-1 heparan sulfate from P3. Analysis of disaccharide composition and oligosaccharide mapping also reveals differences between MPC-11 and P3 heparan sulfate. Most notably, the level of N-sulfation and 2-O-sulfation is higher, and 6-O-sulfation lower, in syndecan-1 heparan sulfate from MPC-11 than from P3. Interestingly, levels of total sulfation of syndecan-1 heparan sulfate from MPC-11 and P3 are similar (75.6 and 72.6 sulfates/100 disaccharides, respectively), indicating that the difference in their affinity for collagen is not due to a difference in net charge. These data indicate that the fine structure of heparan sulfate can differ on identical proteoglycan core proteins, and these differences can control fundamental cellular properties such as cell-matrix adhesion.

Animals↗
PubMed1994

Liver heparan sulfate structure. A novel molecular design.

The structure of rat liver heparan sulfate (HS) has been investigated using a combination of (a) chain scission with specific reagents, (b) disaccharide compositional analysis, and (c) end-referenced sequence analysis of the proximal, protein-linked region of the chain. This study reveals that the liver synthesizes a highly sulfated HS species (1.34 sulfates/disaccharide), particularly high in N-sulfation (60%) and 2-O-sulfate content (36%). Approximately half of the latter is found in trisulfated disaccharides, i.e. IdceA(2-OSO3) alpha 1-4GlcNSO3 (6-OSO3). End-referencing methodology established the existence of an extended, unmodified heparan (GlcUA beta 1-4GlcNAc) sequence, 8-11 disaccharides in length, attached to the linkage tetrasaccharide, similar to that found in a number of other HS species. Directly following this is a mixed HexUA1-4GlcNR(6-OSO3) (where GlcNR represents alpha-D-glucosamine with an unspecified N-substituent)-containing sequence of variable length, culminating in the appearance of the first IdceA(2-OSO3) residue approximately 20 disaccharides from the linkage region, i.e. approximately 40% along the length of the chain. The distal 60% of the polysaccharide is highly sulfated (approximately 2 sulfates/disaccharide) and mainly comprises three heparin-like domains, highly enriched in IdceA(2-OSO3) residues. Overall, liver HS qualifies as an extreme member of the HS family, with a considerable proportion of heparin-like structure asymmetrically concentrated to the distal part of the chain.

Animals↗
PubMed1994

Interaction of hepatocyte growth factor with heparan sulfate. Elucidation of the major heparan sulfate structural determinants.

We have demonstrated by affinity chromatography that hepatocyte growth factor (HGF) binds strongly to heparan sulfate (HS). This substantiates previous suggestions that cell-surface heparan sulfate proteoglycans constitute the so-called low affinity cellular binding sites for HGF. Using a recombinant human HGF affinity column, we have analyzed the effects of various specific chemical and enzymatic modifications/depolymerizations of HS on its affinity in order to elucidate the polysaccharide structural determinants. Interaction is shown to be only slightly affected by digestion with heparinase I or III or by replacement of N-sulfates with N-acetyl groups. This suggests a specific role for sulfated domains containing nonsulfated IdceA residues, with only a small contribution from N-sulfates and IdceA(2-OSO3) residues. In addition, disaccharide analyses of various HGF-binding oligosaccharides indicate that affinity is more closely associated with 6-O-sulfation of GlcNSO3 residues than with sulfation at any other position. Although interaction can be demonstrated with heparinase III-resistant oligosaccharides as small as hexasaccharides, the highest affinity was found with oligosaccharides containing a minimum of 10-12 monosaccharides. The structural specificity of the HGF-HS interaction is thus shown to be radically different from that previously described for the basic fibroblast growth factor-HS interaction.

Animals↗
PubMed1994

Specific heparan sulfate saccharides mediate the activity of basic fibroblast growth factor.

In a previous study, we showed that heparitinase releases a 14-saccharide sequence (Oligo-H) from heparan sulfate (HS) with the structure delta GlcUA beta 1,4GlcNSO3-alpha 1,4[IdceA(2S)alpha 1,4GlcNSO3]5 alpha 1,4IdceA alpha 1,4GlcNAc (where IdceA(2S) represents iduronic acid 2-sulfate), which binds to basic fibroblast growth factor (bFGF) with high affinity (Turnbull, J. E., Fernig, D., Ke, Y., Wilkinson, M. C. & Gallagher, J. T. (1992) J. Biol. Chem. 267, 10337-10341). This paper describes further work on the binding properties of HS saccharides and their capacity to mediate bFGF activity in a mitogenesis assay in which responsiveness is dependent on the addition of HS or heparin. Saccharides prepared by heparinase or nitrous acid digestion and heparitinase-resistant fragments five disaccharide units (degree of polymerization (dp) = 10) or less in size were unable to activate bFGF. However, heparitinase-resistant saccharides of dp12-16 were active in the assay; the dp14 and dp16 fractions were equivalent in activity to heparin and more active than the parent HS. Saccharides of the same size and basic structure as the active fractions (> or = dp12) bound to bFGF with high relative affinity. Active saccharides were composed mainly of N-sulfated disaccharides, the predominant unit being IdceA(2S)-GlcNSO3. This was enriched at least 5-fold in the active saccharides by comparison with the original HS. In addition, the dp12 and dp14 active fractions had a notably low content of trisulfated disaccharides (IdceA(2S)-GlcNSO3(6S)) (where GlcNSO3(6S) represents N-sulfated glucosamine 6-sulfate), which are the major repeat units of heparin. The data show that sequences similar in size and basic structure to Oligo-H can mediate the mitogenic activity of bFGF. Overall, the results provide further evidence that specific HS sequences are generated biosynthetically in order to fulfill particular biological functions such as activation of bFGF.

3T3 Cells↗
PubMed1994

Fibroblast growth factors and their receptors: an information network controlling tissue growth, morphogenesis and repair.

The stimulation of cellular metabolism by the nine fibroblast growth factors (FGFs) is mediated by a dual-receptor system. This comprises a family of four receptor tyrosine kinases (FGFR) and heparan sulphate proteoglycans (HSPG). The stimulation of cell division by FGFs has an obligate requirement for both partners of the dual-receptor system. The binding of the nine FGFs to the FGFRs is marked by a pattern of overlapping specificity despite alternative splicing events generating a large number of FGFR proteins. Thus many of the FGFR isoforms bind several FGFs. It is likely that each FGF requires a different pattern of sulphation within the heparan sulphate chains for binding. Therefore, the HSPG receptors may provide additional specificity, allowing a cell to fine tune its response to the FGFs present in the extracellular milieu. The HSPG receptors also control the availability of FGFs and hence regulate the transport of FGFs within a tissue. FGF-stimulated cell division would appear to have a mandatory requirement for the FGFs to be translocated to the nucleus via the cytosol after interacting with the dual-receptor system. The consequences of the potential direct action of FGFs in stimulating cell division are examined in the light of current models of signal transduction.

Animals↗
PubMed1994

Heparan sulphates as membrane receptors for the fibroblast growth factors.

The complex polymeric structure of heparan sulphate contains intra-chain sequences of sulphated sugar residues that define active sites for the binding and activation of growth factors. The molecular mechanisms of recognition and activation are slowly being revealed at least in the case of the interaction of heparan sulphate with basic fibroblast growth factor. Current data indicate that relatively long but specific binding sequences in heparan sulphate may induce a conformational change in basic fibroblast growth factor exposing a site on the protein that is recognised by signal transducing receptors. Heparan sulphate may also subserve functions of dimerisation of basic fibroblast growth factor and facilitation of receptor transfer by a secondary interaction with the receptor itself. Various models for heparan sulphate mediated induction of mitogenesis by basic fibroblast growth factor have been proposed and there are suggestions that the core protein of plasma membrane heparan sulphate-proteoglycans may participate in the cell signalling process. The vital importance of heparan sulphate in controlling growth factor activities has opened up a new chapter in proteoglycan research and has brought proteoglycans into the mainstream of cell biology. Further investigation of their mode of action is likely to reveal new information on the control of cell growth and development in both embryonic and adult tissues and may suggest novel methods of controlling diseases such as cancer, atherosclerosis or fibrosis that are driven by abnormal expression of growth factors or their receptors.

Animals↗
PubMed1992

Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate.

The structural properties of fibroblast heparan sulfate (HS) that are necessary for it to bind strongly to basic fibroblast growth factor (bFGF) have been investigated using bFGF affinity chromatography. Specific enzymic and chemical scission of HS, together with chemical N-desulfation, revealed that N-sulfate groups and iduronate-2-sulfates (IdoA(2-OSO3)) were essential for the interaction. bFGF-affinity chromatography of sulfated oligosaccharides released from HS by treatment with heparitinase led to the identification of an oligosaccharide component (oligo-H), seven disaccharides in length, with a similar affinity for bFGF as the parent molecule. Heparinase treatment of this fraction abolished the high affinity binding to bFGF. Analysis of oligo-H indicated that 74% of the disaccharide units had the structure IdoA(2-OSO3)alpha 1,4GlcNSO3; the remainder comprised N-acetylated and N-sulfated units, the majority of which were devoid of O-sulfate groups. Oligo-H was fully degraded to disaccharides by treatment with nitrous acid. These results indicate that the sequence of oligo-H is as shown below. delta GlcA beta 1,4GlcNSO3 alpha 1,4[IdoA(2-OSO3)alpha 1,4GlcNSO3]5 alpha 1, 4IdoA alpha 1,4GlcNAc Sulfated oligosaccharides of similar size but with a lower affinity for bFGF had a reduced concentration of IdoA(2-OSO3) but significant quantities of GlcNSO3(6-OSO3) and GlcNAc(6-OSO3). The data indicate a primary role for contiguous sequences of IdoA(2-OSO3)alpha 1,4GlcNSO3 in mediating the high affinity binding between fibroblast HS and bFGF.

Binding Sites↗
PubMed1992

Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver.

We have used an antiserum raised against a purified heparan sulfate proteoglycan (HSPG) preparation isolated from rat liver to screen a lambda gt11 expression library and have obtained overlapping cDNA clones that contain the full-length coding sequence of an HSPG core protein capable of spanning the plasma membrane. The open reading frame of the rat cDNA encodes a protein of 211 amino acids. The predicted protein sequence (23 kDa) has a high degree of homology with the published partial sequence of the human lung fibroblast HSPG, fibroglycan. The deduced protein sequence contains a 24-amino acid transmembrane domain and a 33-amino acid cytoplasmic domain, both of which are identical with the corresponding regions of human fibroglycan and are highly homologous to the human, hamster, and mouse epithelial HSPG, syndecan. The putative ectodomain, which has 85% homology to fibroglycan, contains three possible glycosaminoglycan attachment sites that may be occupied by heparan sulfate chains. The major 49-kDa core protein in the liver HSPG preparation was found to be reactive to an antibody that specifically recognizes the cytoplasmic domain of fibroglycan. We have used the full-length cDNA clone to analyze the expression of this transmembrane core protein gene in whole tissues and several epithelial and fibroblastoid cell lines. It hybridizes to three mRNA species in all cell and tissue types examined, but in liver, isolated hepatocytes, and kidney, an additional 0.8-kilobase mRNA was detected. The three common messages arise from differential use of alternative polyadenylation sites, whereas the fourth tissue-restricted RNA species represents a related gene transcript. The rat equivalent of human fibroglycan therefore appears to be the major transmembrane proteoglycan in liver, and its widespread expression in many diverse tissues and cells suggests that it plays an important role in cellular interactions.

Amino Acid Sequence↗
PubMed1992

Patterns of sulphation in heparan sulphate: polymorphism based on a common structural theme.

HS appears to be a well-organised molecule with a domain structure that is apparently unique amongst the GAG family (Gallagher, 1989). Further refinements in sequence analysis are needed to corroborate the simplified model proposed in Fig. 4. It is still not clear why evolution has favoured a structural motif of widely spaced sulphated domains. Presumably, some advantages must accrue to the organism from this design, and one idea, that we have discussed previously, is that the polysaccharide functions as a "template" for the organisation of structural proteins in the ECM and for the binding and presentation of growth factors within the matrix polymer network. The sulphated regions are likely to display considerable conformational versatility as a result of the presence of the iduronate residues, and this property may be very important for the protein-binding properties of the polysaccharides (Casu et al., 1988). Sulphation patterns within these regions could favour oligosaccharide conformations necessary for specific protein interactions. An important question in this context is why different cells express on their surfaces HS with subtle differences in sulphation pattern. Perhaps the polymorphic features of HS are involved in higher-order tissue- and organ-specific mechanisms controlling cellular recognition and morphogenesis. The consistency with which aberrant sulphation of HS is detected in malignant disease (Gallagher and Lyon, 1989) in which cellular recognition and differentiation are impaired, adds some substance to this view.

Amino Acid Sequence↗
PubMed1992

Cell adhesion in the stromal regulation of haemopoiesis.

Haemopoiesis occurs in close physical contact with elements of the bone marrow stroma. This review examines some adhesion molecules which are expressed in haemopoietic environments. These molecules may act to maintain cell contact between haemopoietic progenitors and the cellular and extracellular matrix elements of the marrow stroma. A review is undertaken of several families of adhesion molecules and their ligands, including integrins, CD44, LEC-CAMs, CAMs and variant glycosylation patterns expressed by haemopoietic cells. Both CD44 and integrin receptors have been implicated in mediating important cellular interactions within the haemopoietic system and the significance of these and other molecules in haemopoiesis is discussed.

Amino Acid Sequence↗