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  • 1
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column–mediated affinity capture of a set of ...
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-0878
    Keywords: Key words: β-Galactoside-binding lectin ; Dermis ; Skin ; Chick embryo ; Immunohistochemistry ; Keratinization ; Mucous metaplasia ; Domestic fowl
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of β-galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light- microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.
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  • 3
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 3 (1990), S. 204-207 
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A characteristic property of anhydrotrypsin, i.e., its ability to strongly bind C-terminal arginine, proved to be useful as a tool for specific enrichment of a recombinant protein. An arginine tail was introduced at the C-terminus, for example, of a human β-galactoside-binding lectin by site-directed mutagenesis. The resulting mutant recombinant lectin, which retained sugar-binding activity as high as the wild-type lectin, became recognizable by anhydrotrypsin. It was adsorbed on an anhydrotrypsin-agarose column and eluted with benzoylglycylarginine. The added arginine tail could be specifically removed by carboxypeptidase B. When E. coli lyzate containing the mutant lectin was applied to the column more than 10-fold enrichment of the mutant lectin was attained. This procedure should be generally applicable and may be advantageous because the addition of a single arginine residue may have minimal effect on the structure and function of the target protein.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 134-140 
    ISSN: 0952-3499
    Keywords: affinophoresis ; affinophore ; affinity probe capillary electrophoresis ; pea lectin ; concanavalin A ; laser-induced fluorescence detection ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Combination of capillary electrophoresis and bioaffinity interaction gave rise to powerful research tools for analyzing molecular recognition. They take advantages of the electrophoretic behavior of the complex formed between a target biomolecule and a specially designed mobile ligand molecule (affinophore or affinity probe), and enable detection of complex formation, determination of the equilibrium constants and stoichiometry, etc. Copyright © 1998 John Wiley & Sons, Ltd.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-4986
    Keywords: β-galactoside-binding lectin ; cell calcium signalling ; Jurkat T cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The effects of the β-galactoside-binding lectin from human placenta (HPL14) on intracellular calcium concentration ([Ca2+]i) were examined in the human Jurkat T cell line. The lectin induces a concentration dependent increase in [Ca2+]i. This calcium signalling effect is clearly mediated through complementary cell surface galactoglycoconjugates because it can be blocked by β-galactosides. The observed Ca2+-response involves both the release of calcium from intracellular stores and a calcium influx from the extracellular space. It is sustained in the presence of 1 mM extracellular calcium whereas it becomes transient when the influx of extracellular calcium was blocked by calcium chelation to EGTA. Voltage-sensitive calcium channel blockers like verapamil and prenylamine were without effect on the action of HPL14. Protection of the sugar binding activity of HPL14 in the absence of a thiol-reducing reagent by carboxamidomethylation (CM-HPL14) or by substitution Cys2 with serine (C2S) results in lectin proteins with considerably decreased calcium signalling efficiency. The recombinant lectin (Rec H) and the mutant protein obtained by substitution of highly conservative Trp68 with tyrosine (W68Y) induce lower levels of [Ca2+]i compared to wild type lectin.
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  • 6
    ISSN: 1573-4986
    Keywords: galectin ; mutagenesis ; carbohydrate-recognition domain
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract To identify critical amino acid residues for carbohydrate binding of galectins (soluble β-galactoside-binding lectins found in the animal kingdom), site-directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992)J Biol Chem 266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate-binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate-binding site of galectin.
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  • 7
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The localization of the 32-kDa galectin (β-galactoside-binding lectin) of the nematodeCaenorhabditis elegans, which is the first lectin to be found in a nematode, was examined immunohistochemically using an anti-lectin antiserum. The lectin was found to be localized most abundantly in the adult cuticle and also in the terminal bulb of the pharynx. However, it was difficult to locate the galectin in larval animals, though immunochemical experiments suggested its presence. These results suggest that one of the fundamental roles of the galectin may be as a component of the durable outer barrier, as in the case of the morphogenesis of chick embryonic skin.
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  • 8
    ISSN: 1432-0878
    Keywords: Key words: Galectin ; β-Galactoside-binding lectin ; Human ; Skin ; Immunocytochemistry ; Immunohistochemistry ; Hybridization ; in situ ; Langerhans cell ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The localization of an endogenous 14-kDa β-galactoside-binding lectin (galectin) and its pattern of gene expression were examined in normal human skin by light- and electron microscopy. Under the light microscope, immunostaining of 14-kDa galectin was observed in the cell membrane of cells in the basal and spinous layers of the epidermis. Galectin was also found in the Langerhans cells, as shown by double labeling using anti-14-kDa galectin and anti-CD1a antibodi es. In the dermis, immunostaining for the 14-kDa galectin was positive in the extracellular matrix and fibroblasts. At the electron-microscopic level of resolution, galectin was located primarily along the plasma membrane of keratinocytes, and in both the cytoplasm and nucleus of Langerhans cells in the epidermis, whereas in the dermis it was detected in the extracellular matrix and in both the nucleus and cytoplasm of fibroblasts. The gene expression of 14-kDa galectin was visualized by the HRP-staining me thod following in situ hybridization techniques. The expression was detected in the cytoplasm of cells in the basal and spinous layers of the epidermis; whereas, in the dermis, it was detected in the cytoplasm of fibroblasts. Moreover, SDS-polyacrylamide gel electrophoresis and lectin-blot analysis revealed that this galectin bound to glycoproteins of approximately 17, 62, and 72 kDa in the epidermis and to those of 29, 54, and 220 kDa in the dermis. The present study indicates that 1) normal human skin produces the β-galactoside-binding 14-kDa galectin, and 2) this galectin is located in both the epidermis, particularly in the keratinocytes and Langerhans cells, and in the dermis. These results suggest that galectin is important for cell-cell contact and/or adhesion in the epidermis and for cell-extracellular matrix interaction in the dermis.
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  • 9
    ISSN: 1432-0878
    Keywords: β-Galactoside-binding lectin ; Dermis ; Skin ; Chick embryo ; Immunohistochemistry ; Keratinization ; Mucous metaplasia ; Domestic fowl
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of β-galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.
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  • 10
    ISSN: 1432-0878
    Keywords: Galectin ; β-Galactoside-binding lectin ; Human ; Skin ; Immunocytochemistry ; Immunohistochemistry ; Hybridization, in situ ; Langerhans cell ; Man
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The localization of an endogenous 14-kDa β-galactoside-binding lectin (galectin) and its pattern of gene expression were examined in normal human skin by light- and electron microscopy. Under the light microscope, immunostaining of 14-kDa galectin was observed in the cell membrane of cells in the basal and spinous layers of the epidermis. Galectin was also found in the Langerhans cells, as shown by double labeling using anti-14-kDa galectin and anti-CD1a antibodies. In the dermis, immunostaining for the 14-kDa galectin was positive in the extracellular matrix and fibroblasts. At the electron-microscopic level of resolution, galectin was located primarily along the plasma membrane of keratinocytes, and in both the cytoplasm and nucleus of Langerhans cells in the epidermis, whereas in the dermis it was detected in the extracellular matrix and in both the nucleus and cytoplasm of fibroblasts. The gene expression of 14-kDa galectin was visualized by the HRP-staining method following in situ hybridization techniques. The expression was detected in the cytoplasm of cells in the basal and spinous layers of the epidermis; whereas, in the dermis, it was detected in the cytoplasm of fibroblasts. Moreover, SDS-polyacrylamide gel electrophoresis and lectin-blot analysis revealed that this galectin bound to glycoproteins of approximately 17, 62, and 72 kDa in the epidermis and to those of 29, 54, and 220 kDa in the dermis. The present study indicates that 1) normal human skin produces the β-galactoside-binding 14-kDa galectin, and 2) this galectin is located in both the epidermis, particularly in the keratinocytes and Langerhans cells, and in the dermis. These results suggest that galectin is important for cell-cell contact and/or adhesion in the epidermis and for cell-extracellular matrix interaction in the dermis.
    Type of Medium: Electronic Resource
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