Biomedical subjects

R C Hughes

Publications and source records attributed to R C Hughes.

At least 37 records · Page 2Linked to original sources

PubMed1997

Detection and distribution of the carbohydrate binding protein galectin-3 in human notochord, intervertebral disc and chordoma.

Galectin-3 is an endogenous carbohydrate-binding protein which plays a role in cell differentiation, morphogenesis and cancer biology. We investigated the occurrence and distribution of galectin-3 in the embryonic and fetal human notochord, the developing human vertebral column, adult intervertebral discs and in six chordomas, which are tumors thought to originate from notochordal remnants. By means of Western blots, the expression of galectin-3 was confirmed in tissue probes from the vertebral column region beginning with the 8th gestational week. These results were supported by immunohistochemical data which revealed the presence of galectin-3 in the cytoplasm of cells of the notochord also from the 8th gestational week onwards. Notochordal immunostaining became stronger with increasing gestational age. A persisting notochordal remnant in an adult intervertebral disc and various cells of the nucleus pulposus also contained galectin-3. All chordomas showed moderate or strong immunoreactivity irrespective of their cellular composition. Subcellularly, galectin-3 was localized mostly in the cytoplasm, while a subset of tumor cells also showed nuclear distribution. Differences in staining patterns of chordoma cells could not, in general, be correlated to any histological features of these tumors.

Adolescent↗

PubMed1997

Plasma membrane targetting, vesicular budding and release of galectin 3 from the cytoplasm of mammalian cells during secretion.

Galectin 3, a 30 kDa galactoside-binding protein distributed widely in epithelial and immune cells, contains no signal sequence and is externalized by a mechanism independent of the endoplasmic reticulum (ER)-Golgi complex. We show here that hamster galectin 3 overexpressed in transfected cos-7 cells is secreted at a very low rate. A chimaera of galectin 3 fused to the N-terminal acylation sequence of protein tyrosine kinase p56(lck), Nt-p56(lck)-galectin 3, which is myristoylated and palmitoylated and rapidly transported to plasma membrane domains, is efficiently released from transfected cells indicating that movement of cytoplasmic galectin 3 to plasma membrane domains is a rate limiting step in lectin secretion. N-terminal acylation is not sufficient for protein secretion since p56(lck) and the chimaera Nt-p56(lck)-CAT are not secreted from transfected cells. The amino-terminal half of galectin 3 is sufficient to direct export of a chimaeric CAT protein indicating that part of the signal for plasma membrane translocation lies in the N-terminal domains of the lectin. Immunofluorescence studies show that Nt-p56(lck)-galectin 3 aggregates underneath the plasma membrane and is released by membrane blebbing. Vesicles of low buoyant density isolated from conditioned medium are enriched in galectin 3. The lectin is initially protected from exogenous collagenase but is later released in soluble protease-sensitive form from the lectin-loaded vesicles. Using murine macrophages, which secrete their endogenous galectin 3 at a moderate rate especially in the presence of Ca2+-ionophores, we were also able to trap a galectin 3-loaded vesicular fraction which was released into the culture supernatant.

Acylation↗

PubMed1997

Epithelial galectin-3 during human nephrogenesis and childhood cystic diseases.

Galectin-3 is a beta-galactoside-binding protein with putative roles in development, oncogenesis, and inflammation. Its expression in human nephrogenesis has not been previously reported. This study examines galectin-3 expression in early human embryos by Western blot and immunohistochemistry. This 33-kD protein was detected in the apical domain of distal tubules of the mesonephros and also in the mesonephric duct. In the metanephros, the adult kidney precursor, galectin-3 was detected in the apical domains of ureteric bud branches, and there was intense expression in fetal medullary and papillary collecting ducts in both the cytoplasm and plasma membranes. Low levels of galectin-3 were detected in the cytoplasm of a subset of cells in adult collecting ducts; these were alpha-intercalated cells because they expressed basal band 3 protein. In human multicystic dysplastic kidneys, all diseased epithelia had an embryonic apical expression pattern of galectin-3 and, in addition, all cystic epithelia in autosomal recessive polycystic kidneys expressed this molecule. It is concluded that galectin-3 is expressed by cells of the mesonephric duct/ureteric bud lineage, and it is speculated that the different subcellular locations may be implicated in both the regulation of normal growth and differentiation of this lineage, as well as in the pathogenesis of cystic epithelia.

Adult↗

PubMed1996

Galectin-3 stimulates uptake of extracellular Ca2+ in human Jurkat T-cells.

Galectin-3, a mammalian galactoside-binding protein, is not expressed in the Jurkat T-lymphoblastoid cell line. However, Jurkat cells express surface glycoprotein receptors for galectin-3, one of which is shown to be the glycosylated heavy chain of CD98 (4F2 antigen), a T-cell activation marker. Addition of galectin-3 to Jurkat cells triggers a sustained influx of extracellular Ca2+ in a concentration dependent manner. The induced increase in cytosolic [Ca2+]i is blocked by sugar hapten inhibitors of galectin-3. The galectin-3-induced effect is insensitive to voltage-gated Ca2+ channel antagonists such as prenylamine, nifedipine and diltiazem and to pertussis toxin but is inhibited by cholera toxin. The results suggest that galectin-3 released by accessory cells such as macrophages may bind in vivo to T-cell activation antigens and also participate in Ca2+ signalling.

Animals↗

PubMed1996

Immunocytochemical evidence for a modulation of galectin 3 (Mac-2), a carbohydrate binding protein, in pulmonary fibrosis.

Galectin 3 is endogenous mammalian carbohydrate-binding protein with affinity for terminal beta-galactose residues, polylactosamine glycans, and ABH-blood group carbohydrate epitopes. To determine the distribution and regulation of galectin 3 during pulmonary injury, which is known to be accompanied by profound changes in the carbohydrate moieties of cell surface glycoproteins of alveolar cells, a rat model of irradiation-induced lung inflammation and repair was used. Immunocytochemistry showed that in normal rat lungs, galectin 3 was localized to alveolar macrophages, with weaker staining of bronchial epithelial cells. Shortly after irradiation-induced lung injury, when there is active proliferation of type II alveolar epithelial cells and re-epithelialization of alveolar basement membranes by type I cells, the total galectin concentration in the lung increased dramatically. This increase was due in part to an increased population of galectin 3-positive interstitial and alveolar macrophages. In addition, galectin 3 was expressed prominently at the surface of the newly formed type I alveolar epithelium and to lesser extent at the apical surface of type II cells. These findings suggest that the increased synthesis and secretion of galectin 3 during irradiation-induced lung injury, together with ligation of secreted lectin at the surface of alveolar epithelial cells, may play roles in pulmonary alveolar epithelial expansion and differentiation during injury and repair.

Animals↗

PubMed1996

Effects of the carbohydrate-binding protein galectin-3 on the invasiveness of human breast carcinoma cells.

Galectin-3 is a Mr 30,000 protein with carbohydrate-binding specificity for type I and II ABH blood group epitopes and polylactosamine glycans expressed on cell surface and extracellular matrix glycoproteins such as laminin. Cell lines propagated from human normal mammary epithelia and from benign or infiltrating components of primary breast tumours express low levels of galectin-3 in the cytoplasm. However, galectin-3 when added exogenously in solution or when bound within a three-dimensional matrix markedly enhanced the migration of the primary tumour cell lines through a Matrigel barrier. Galectin-3 expression in the cytoplasm and intercellularly on surface membranes was greatly increased in cell lines propagated from malignant ascites and pleural effusions of late stage breast cancer. These cell lines were non-invasive in the Matrigel assay and exogenous galectin-3 had no enhancing effect on invasiveness. These results suggest that galectin-3 could play multiple roles in cell metastasis at an early invasive stage by acting in a paracrine manner to stimulate cell migration through an extracellular matrix, and in later stage cancers in synergy with other mediators of cell-cell aggregation. However, endogenous galectin-3 expression in human breast cancers is not correlated directly with their invasive potential in vitro.

Antigens, Differentiation↗

PubMed1995

Cross-linking of galectin 3, a galactose-binding protein of mammalian cells, by tissue-type transglutaminase.

The 30 kDa beta-galactoside-binding protein of baby hamster kidney (BHK) cells [Mehul et al. (1994), J. Biol. Chem. 269, 18250-18258] homologous to galectin 3, a widely distributed mammalian lectin, has been found to be a substrate for tissue type transglutaminase, as shown by the incorporation in a calcium- and time-dependent manner of 5-(biotinamido) pentylamine in the presence of guinea pig liver transglutaminase. The amino-terminal domain of hamster galectin 3, which is a repetitive sequence rich in glutamine, tyrosine, glycine and proline, is also an excellent substrate. A single lysine residue in the N-terminal domain is an essential requirement for transglutaminase-mediated oligomerization, and two equivalent glutamine residues present in identical sequence repeats within this domain appear to be involved as amine acceptors in cross-linking reactions. Transglutaminase-mediated cross-linking of galectin 3 to itself or to matrix components may be one mechanism for stabilisation of a multivalent binding form of the lectin in cell secretions or in extracellular matrices.

Amino Acid Sequence↗

PubMed1995

Galectin-3 expression and effects on cyst enlargement and tubulogenesis in kidney epithelial MDCK cells cultured in three-dimensional matrices in vitro.

Galectin-3 is a member of a closely related family of beta-galactoside-binding soluble proteins found in many vertebrate epithelial and myeloid cell types. The developmentally regulated presence of galectin-3 in tissues, for example kidney, and an affinity for many cell-surface and matrix glycoproteins indicate its importance in extracellular biological processes. Since a polarised expression and secretion of galectin-3 was observed in monolayer-cultured MDCK cells, an understanding of the secretion and distribution of this lectin in a three-dimensional in vitro model would help to uncover its role(s) in the interplay between cell-surface adhesion molecules and extracellular matrix components occurring during cell aggregation and polarisation in tissue formation. In this study, the cellular distribution and secretion of galectin-3 were examined in MDCK cells cultured within a gel matrix. MDCK cells were cultured within type I collagen or Matrigel to obtain multicellular cysts, and tubule formation was induced in collagen gels with hepatocyte growth factor. Immunofluorescent staining of these structures using antibodies against galectin-3 and other cell-surface domain markers was carried out either in situ or on cryosections and was visualised by confocal and conventional epifluorescence microscopy. Our results show that MDCK cells suspended in hydrated collagen gels or Matrigel exhibit differential and polarised galectin-3 expression on the baso-lateral surface domains of cells lining the cysts. The lectin is colocalised with laminin on the basal surface. In tubule-forming cysts, galectin-3 is excluded from the initial spikes and the progressing tips of the tubules although its basolateral expression on the cyst body remains. Galectin-3 added exogenously to cultures, as well as antibodies against laminin subunits and integrin beta 1 subunit, exerted an inhibitory effect on cyst enlargement of MDCK cells in 3-D Matrigel while galectin-3-specific antibodies could promote this process. The results suggest that galectin-3 exerts its effect on MDCK cells in a three-dimensional environment through modulation of both cell-cell and cell-substratum adhesions, and the interplay between these adhesions is important in the growth of multicellular aggregates and extensions occurring during normal kidney tubulogenesis.

Animals↗

PubMed1994

Structure of baby hamster kidney carbohydrate-binding protein CBP30, an S-type animal lectin.

A galactose-binding protein of M(r) = 30,000 previously described in baby hamster kidney cells (Foddy, L., Stamatoglou, S. C., and Hughes, R. C. (1990) J. Cell Sci. 97, 139-148) has been analyzed by the cloning and sequencing of cDNA clones encoding the complete sequence and an amino-terminal fragment. The intact lectin CBP30 contains 245 amino acid residues, including the initiating methionine residue, and is closely homologous to mammalian S-type lectins of similar size characterized in human, rat, and mouse species. The carboxyl-terminal domain contains the carbohydrate binding activity and the amino-terminal domain, which is extremely sensitive to bacterial collagenase, contains a repetitive sequence rich in glycine, tyrosine, and proline. There are 8 repeats in hamster CBP30, as in the human homologue, compared with about 10 in rat and mouse and > 10 in dog homologues. This repeat sequence is also sensitive to the tissue metalloproteinases, gelatinase B and matrilysin, but, unlike the bacterial collagenase, the mammalian enzymes also cause extensive degradation of the carbohydrate binding carboxyl domain. Physical measurements using CD and tryptophan fluorescence spectroscopy indicate that the two domains of CBP30 are structurally, as well as functionally, distinct and independent. Cross-linking studies indicate that the amino-terminal lectin fragment can efficiently self-assemble into oligomeric species, and less efficient but significant aggregation of the intact lectin is also shown. Domain-specific antibodies to hamster CBP30 have been prepared and used to show that only the full-length, undegraded form of CBP30 is present in whole cell lysates.

Amino Acid Sequence↗

PubMed1994

Cell adhesion molecules in liver function and pattern formation.

In epithelia, adhesive interactions participate in specific histogenetic events and also instate a morphological polarization that is linked to the vectorial functioning of the epithelium. Cell polarity is a recurring event in differentiation, first observed in the preimplantation embryo and then during organogenesis, whereas in oncogenesis a breakdown of polarity may occur. We review here recent work on adhesive interactions in the liver parenchyma. We focus on the effect of cell-biomatrix interactions on hepatocyte function and polarization, on the integration of adhesive cell-cell and cell-matrix interactions, and on the correlation between structural differentiation and adhesion during hepatic development, regeneration, and oncogenesis. Although highlighting morphological differences between the liver and other epithelia, we stress that adhesion-mediated effects such as substratum regulation of differentiation, feedback control of matrix synthesis, and integration of intercellular and cell-matrix interactions, shown to transpire in the liver, most probably occur in other parenchymal tissues too.

Animals↗

PubMed1994

Regulation of secretion and surface expression of Mac-2, a galactoside-binding protein of macrophages.

Mac-2, a 30-35-kDa galactose-binding protein, is synthesized at similar levels in murine peritoneal exudate macrophages whether recruited in response to an intraperitoneal pathogen Mycobacterium microti, to sterile inflammatory stimuli such as thioglycollate broth, or to concanavalin A. In elicited or activated macrophages up to 30% of Mac-2 is constitutively secreted, and secretion is stimulated markedly by calcium ionophore A23187. Only thioglycollate-elicited macrophages express cell surface Mac-2, and binding is mostly (> 80%) a result of affinity for cell surface carbohydrate structures. Mac-2 surface expression is markedly reduced upon further activation of thioglycollate-elicited macrophages with bacterial lipopolysaccharide in vitro. Polylactosamine structures are present on all macrophage populations examined as determined by binding of Lycopersicon esculentum lectin, whereas alpha-galactosyl residues detected by Griffonia simplicifolia isolectin B4 are expressed only on the thioglycollate-elicited macrophages, indicating that these residues are the major determinants responsible for Mac-2 surface expression. Chemical cross-linking experiments have identified binding of endogenous cell-surface Mac-2 to three glycoproteins of molecular masses of 92, 125, and 180 kDa containing alpha-galactosyl and polylactosamine structures on thioglycollate-elicited macrophages. The restricted cell surface distribution of Mac-2 on thioglycollate-elicited peritoneal macrophages, a population of recently recruited monocytes, suggests a role(s) in early events of macrophage infiltration and tissue fixation such as extravasion and cell-matrix interactions.

Animals↗

PubMed1994

Control of Mac-2 surface expression on murine macrophage cell lines.

Mac-2 antigen, a 32-kDa murine macrophage cell-surface protein expressed on thioglycollate-elicited peritoneal exudate cells at higher levels than other macrophages, is a member of the S-(soluble) galactoside-binding lectin family with homologies to carbohydrate-binding proteins of other cell types. Murine macrophage cell lines can be ordered in a linear differentiation sequence according to their expression of Mac-2 and other surface markers (Leenen et al., Differentiation 1986. 32: 157.) We show here that antigen expression in macrophage cell lines can be regulated at the level of protein secretion. WEHI-3 cells, classified as immature macrophages by virtue of their low level of surface Mac-2 expression synthesize similar amounts of the antigen as more mature J774.2 and P388.D1 cells that express high amounts of surface Mac-2, but unlike these latter cell lines WEHI-3 cells fail to secrete the protein. Exogenously added Mac-2 binds efficiently to WEHI-3 cells and putative Mac-2-binding carbohydrates are expressed equally on WEHI-3, J774.2 and P388.D1 cells as judged by binding of plant lectins of known carbohydrate-binding specificities. Mac-2 secretion and surface expression in WEHI-3 cells is not significantly enhanced by calcium ionophore A23187, a powerful stimulator of Mac-2 secretion in other cells and a moderate stimulator in J774.2 and P388.D1 cells. WEHI-3 cells provide a valuable system for studying the mechanism of intracellular transport and secretion of Mac-2, a protein that lacks a signal sequence and does not enter the classical secretory pathway.

Animals↗

PubMed1994

Characterization of isolectins in Tetracarpidium conophorum seeds (Nigerian walnut).

A lectin preparation obtained from Tetracarpidium conophorum (Nigerian walnut) by affinity chromatography of seed extracts on lactose-agarose has been shown to contain two components by gel filtration on Sephadex G150. The larger component Tetracarpidium conophorum agglutinin I (TCAI) is a disulphide-bonded 70 kDa homodimer whereas the second component TCAII is a 34 kDa monomeric protein. Amino terminal aminoacid sequencing shows identity in TCAI and TCAII for the first fifteen residues after which the sequences diverge. The N-terminal sequences of TCAI and TCAII show identity with sequences in the B-chains of ricin and Ricinus communis agglutinin I (RCAI) in eleven of the initial fifteen residues. Thereafter TCAI appears to be homologous to the ricin B chain whereas TCAII is more homologous with the B chain of RCAI. A limited screening of the carbohydrate-binding specificity of TCAII by affinity chromatography of defined oligosaccharides on TCAII Sepharose columns shows that the binding specificity reported earlier for affinity purified Tetracarpidium conophorum isolectins (Sato S, Animashaun T, Hughes RC (1991) J Biol Chem 266:11485-94) reflects the binding properties of TCAII which is the major isolectin in unfractionated lectin preparations.

Amino Acid Sequence↗

PubMed1994

Mac-2: a versatile galactose-binding protein of mammalian tissues.

Mac-2 is a member of the S-(soluble) lectin family. Its identification and isolation from a wide variety of cell types and tissues suggest a diversity of roles in various biological systems. The key points to be made are that Mac-2, and the S-lectins in general, by virtue of their recognition of a variety of carbohydrate structures expressed on different glycoproteins, are well placed to exert discrete biological effects according to the distribution of those glycoproteins in tissues and their differential patterns of glycosylation according to developmental status and cell type. In this regard, the lectins are fundamentally different in character to other effector molecules that in general bind to specific receptors to trigger single signal transduction events.

Amino Acid Sequence↗

PubMed1993

Bowringia mildbraedii agglutinin: polypeptide composition, primary structure and homologies with other legume lectins.

The amino-acid sequences of the subunits of the lectin BMA from seeds of Bowringia mildbraedii have been determined. The data indicate that the lectin consists of a precursor polypeptide of approx. 29 kDa that is cleaved almost completely into two fragments of approx. 13.3 kDa (alpha subunit) and approx. 11.9 kDa (beta subunit), respectively. The beta subunit represents the N-terminal half of precursor polypeptides and is disulphide-linked in a beta beta dimer in the native (alpha beta)2 protein. BMA shows extensive amino-acid sequence homologies with known legume lectins. The site of post-translational proteolysis of the putative precursor occurs at a position similar to that identified in lectins obtained from other Sophoreae plants such as Sophora japonica and in Diocleae lectins such as Concanavalin A, but different from that of two chain lectins obtained from other tribes of the Papilionaceae.

Agglutinins↗

PubMed1993

Secretion of the baby hamster kidney 30-kDa galactose-binding lectin from polarized and nonpolarized cells: a pathway independent of the endoplasmic reticulum-Golgi complex.

A carbohydrate-binding protein of molecular weight 30 kDa (CBP30) isolated from baby hamster kidney (BHK) cells binds polylactosamine glycans present prominently on extracellular matrix glycoproteins of oncofetal origin, such as Engelbroth-Holm-Swarm (EHS) tumor laminin and amniotic fluid fibronectin, and inhibits attachment and spreading of BHK cells to EHS laminin substrata mediated by integrin(s) suggesting an extracellular function for the lectin (S. Sato and R. C. Hughes (1992) J. Biol. Chem. 267, 6983-6990). Here we show that CBP30 shares amino acid sequence homologies with other lectins of similar size, e.g., murine CBP35, Mac2 antigens, and rat IgE-binding protein. Unlike most secreted proteins these lectins contain no signal sequence and we report that drugs, such as brefeldin A and monensin, which inhibit the intracellular transport of classical secretory (glyco)proteins do not block secretion of CBP30 from BHK cells. Secretion is inhibited by methylamine and serum starvation and is increased by heat shock and calcium ionophore A23187, treatments known to block or stimulate exocytosis, respectively. Immunofluorescence and biochemical analysis shows that CBP30 is distributed throughout the cytoplasm of subconfluent BHK cells where it turns over with a half-life of about 30 h, and small amounts are also deposited on the cell surface and substratum. At confluency, the CBP30 assembles into patches that eventually appear to underlie the plasma membrane and extracellular deposits become more numerous. In filter-grown confluent monolayers of Madin-Darby canine kidney cells the lectin is secreted from and expressed at the apical domain of the polarized cells whereas laminin is secreted from the basal domain and becomes incorporated into the matrix between cells and substratum.

Amino Acid Sequence↗

PubMed1992

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.

1-Deoxynojirimycin↗

PubMed1992

Brain implants in man do not break down the blood-brain barrier to dopamine and domperidone.

We have evaluated the blood-brain barrier (BBB) in 8 Parkinsonian patients before and after stereotactic implantation of foetal mesencephalon (STIM) and one patient with an adrenal medullary implant. Parenteral administration of dopamine did not reverse Parkinsonism pre-operatively or at 5 days, 1, 2, 3, 4 months and 1 year post-operatively. Apomorphine and domperidone reversed Parkinsonism and produced dyskinesia in all patients pre- and post-operatively. We conclude that the BBB remains intact to dopamine following implantation.

Adult↗