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Svend Kirkeby - One of the best experts on this subject based on the ideXlab platform.
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differential binding of Griffonia simplicifolia 1 isolectin b4 gs1b4 to alpha galactose antigen in porcine adrenal gland
Xenotransplantation, 2005Co-Authors: Gitte Christensen, Axel Kornerup Hansen, Svend KirkebyAbstract:Differential binding of Griffonia simplicifolia 1 isolectin B4 (GS1B4) to alpha-galactose antigen in porcine adrenal gland: P6:02 Gitte Christensen;Axel Hansen;Svend Kirkeby; Xenotransplantation
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comparison of the binding properties of the mushroom marasmius oreades lectin and Griffonia simplicifolia i b4 isolectin to αgalactosyl carbohydrate antigens in the surface phase
Xenotransplantation, 2004Co-Authors: Svend Kirkeby, Harry C Winter, Irwin J. GoldsteinAbstract:Kirkeby S, Winter HC, Goldstein IJ. Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B4 isolectin to agalactosyl carbohydrate antigens in the surface phase. Xenotransplantation 2004; 11: 254-261. � Blackwell Munksgaard, 2004 Abstract: The binding of two a-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B4 (GS I-B4 )t o neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various a-galactosylated glycans and laminin from basement membrane of mouse sarcoma that contains the xenogenic Gala1-3Gal1-4GlcNAc epitope were immobilized in microtiter plate wells and lectin binding determined with an enzyme- linked assay. After 24 h of incubation, MOA had higher affinity for the xenogenic pentasaccharide (Gala1-3Gal1-4GlcNAcb1-3Galb1-4Glc) than for the Gala-monosaccharide. The binding properties of MOA and GS I-B4 to the xenogenic disaccharide (Gala1-3Galb1) were comparable while the binding of MOA to the xenogenic pentasaccharide was much stronger than the binding of GS I-B4 to the same epitope. Non-xeno- genic disaccharide-coupled neoglycoproteins having galactose end groups linked a1-2 or a1-4 to Gal or linked a1-3 to GalNAc bound very weakly to MOA, whereas GS I-B4 recognized all of these disac- charides with similarly high affinity. MOA also showed high affinity for laminin. The results indicate that the Marasmius oreades lectin has nearly the same affinities as does GS I-B4 for the simple xenogenic carbohydrate antigens, but MOA has greater affinity for the pentasac- charide and is far more specific in its binding preferences than the Griffonia lectin.
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comparison of the binding properties of the mushroom marasmius oreades lectin and Griffonia simplicifolia i b4 isolectin to αgalactosyl carbohydrate antigens in the surface phase
Xenotransplantation, 2004Co-Authors: Svend Kirkeby, Harry C Winter, Irwin J. GoldsteinAbstract:The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4) (GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and laminin from basement membrane of mouse sarcoma that contains the xenogenic Galalpha1-3Gal1-4GlcNAc epitope were immobilized in microtiter plate wells and lectin binding determined with an enzyme-linked assay. After 24 h of incubation, MOA had higher affinity for the xenogenic pentasaccharide (Galalpha1-3Gal1-4GlcNAcbeta1-3Galbeta1-4Glc) than for the Galalpha-monosaccharide. The binding properties of MOA and GS I-B(4) to the xenogenic disaccharide (Galalpha1-3Galbeta1) were comparable while the binding of MOA to the xenogenic pentasaccharide was much stronger than the binding of GS I-B(4) to the same epitope. Non-xenogenic disaccharide-coupled neoglycoproteins having galactose end groups linked alpha1-2 or alpha1-4 to Gal or linked alpha1-3 to GalNAc bound very weakly to MOA, whereas GS I-B(4) recognized all of these disaccharides with similarly high affinity. MOA also showed high affinity for laminin. The results indicate that the Marasmius oreades lectin has nearly the same affinities as does GS I-B(4) for the simple xenogenic carbohydrate antigens, but MOA has greater affinity for the pentasaccharide and is far more specific in its binding preferences than the Griffonia lectin.
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binding of Griffonia simplicifolia 1 isolectin b4 gs1 b4 to alpha galactose antigens
Immunology and Cell Biology, 2001Co-Authors: Svend Kirkeby, Dennis MoeAbstract:Glycoconjugates with terminal Gal1–3Gal1–4GlcNAc sequences (-galactosyl epitopes, natural xenoreactive antigens) are present on various tissues in pigs and are recognized by human anti-galactosyl (Gal) antibodies1. Hence xenotransplantation (pig-to-human) would trigger immune reactions involving complement activation and lead to the hyperactute rejection of the graft. Xenoreactive antigens are often studied by using the lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4), which shows high affinity to galactose. We here estimate the specificity of GS1 B4 for detecting various galactosyl epitopes by measuring lectin binding to neoglycoproteins, thyroglobulin and pig skeletal muscle. Enzyme linked lectin assays confirmed that GS1 B4 was highly specific to -galactosylated neoglycoproteins while the lectin did not detect a -galactosylated ligand. The length of the sugar chains influenced the lectin–carbohydrate interaction. A monosaccharide linked to serum albumin showed higher lectin affinity than did neoglycoproteins with di- and tri--galactosyl epitopes. When the carbohydrate was extended, as in the xenoreactive pentasaccharide (Gal1-3Gal1-4GlcNAc1-3Gal1-4Glc), the carbohydrate– lectin interaction was meagre. Not only the terminal, but also the subterminal sugar affected the lectin binding because the GS1 B4 affinity to Gal1-3Gal was much stronger than to Gal1-3GalNAc. In bovine and porcine thyroglobulin most Gal epitopes appear to be cryptic, but are unmasked by a heat denaturation. In pig skeletal muscle there was lectin reaction not only in the muscle capillaries, but also in the connective tissue and intracellularly in muscle fibres. In Western blots of isolated proteins from pig muscle at least three bands were strongly stained after incubation with lectin.
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binding of Griffonia simplicifolia 1 isolectin b4 gs1 b4 to α galactose antigens
Immunology and Cell Biology, 2001Co-Authors: Svend KirkebyAbstract:: Glycoconjugates with terminal Galalpha1-3Galbeta1-4GlcNAc sequences (alpha-galactosyl epitopes, natural xenoreactive antigens) are present on various tissues in pigs and are recognized by human anti-alphagalactosyl (alphaGal) antibodies1. Hence xenotransplantation (pig-to-human) would trigger immune reactions involving complement activation and lead to the hyperactute rejection of the graft. Xenoreactive antigens are often studied by using the lectin Griffonia simplicifolia 1 isolectin B4 (GS1 B4), which shows high affinity to galactose. We here estimate the specificity of GS1 B4 for detecting various galactosyl epitopes by measuring lectin binding to neoglycoproteins, thyroglobulin and pig skeletal muscle. Enzyme linked lectin assays confirmed that GS1 B4 was highly specific to alpha-galactosylated neoglycoproteins while the lectin did not detect a beta-galactosylated ligand. The length of the sugar chains influenced the lectin-carbohydrate interaction. A monosaccharide linked to serum albumin showed higher lectin affinity than did neoglycoproteins with di- and tri-alpha-galactosyl epitopes. When the carbohydrate was extended, as in the xenoreactive pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), the carbohydrate- lectin interaction was meagre. Not only the terminal, but also the subterminal sugar affected the lectin binding because the GS1 B4 affinity to Galalpha1-3Gal was much stronger than to Galalpha1-3GalNAc. In bovine and porcine thyroglobulin most alphaGal epitopes appear to be cryptic, but are unmasked by a heat denaturation. In pig skeletal muscle there was lectin reaction not only in the muscle capillaries, but also in the connective tissue and intracellularly in muscle fibres. In Western blots of isolated proteins from pig muscle at least three bands were strongly stained after incubation with lectin.
G. Grant - One of the best experts on this subject based on the ideXlab platform.
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anterograde transport of horseradish peroxidase conjugated isolectin b4 from Griffonia simplicifolia i in spinal primary sensory neurons of the rat
Brain Research, 1998Co-Authors: Fredrik H Wang, Brita Robertson, G. GrantAbstract:Anterograde transport of the isolectin B4 from Griffonia simplicifolia I (B4) conjugated to horseradish peroxidase (HRP) was investigated in rat somatic and visceral primary sensory neurons at different spinal levels. Injection of B4-HRP into the L5 dorsal root ganglion (DRG) resulted in labelling in the sural nerve, but not in the gastrocnemius nerves. Free nerve endings and lanceolate-like nerve endings were labelled in the lateral hindpaw skin. Labelled fibres were also observed in the greater splanchnic nerve following B4-HRP injection into the T10–11 DRGs. Electron microscopic examination of the labelled nerves showed that B4-HRP labelled exclusively unmyelinated axons. In the spinal cord, labelling was observed in the superficial dorsal horn, and additionally, although much more sparse, in the medial and lateral collateral projections following injections into the T10–11 DRGs. The results suggest that B4-HRP should be a suitable anterograde tracer of unmyelinated cutaneous and splanchnic but not muscle primary afferent fibres.
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retrograde and transganglionic transport of horseradish peroxidase conjugated cholera toxin b subunit wheatgerm agglutinin and isolectin b4 from Griffonia simplicifolia i in primary afferent neurons innervating the rat urinary bladder
Neuroscience, 1998Co-Authors: H F Wang, Peter Shortland, M J Park, G. GrantAbstract:In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6–S1 spinal cord segments and in the T13–L2 and L6–S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit–horseradish peroxidase was injected. Cholera toxin B subunit–horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV–VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin–horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit–horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I–horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit–horseradish peroxidase than with wheat germ agglutinin– or isolectin B4–horseradish peroxidase. In the L6–S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with myelinated fibres). Double labelling with other neuronal markers showed that 71%, 43% and 36% of the cholera toxin B subunit-immunoreactive cells were calcitonin gene-related peptide-, isolectin B4-binding- and substance P-positive, respectively. A few cholera toxin B subunit cells showed galanin-immunoreactivity, but none were somatostatin-, vasoactive intestinal polypeptide-, or neuropeptide Y-immunoreactive or contained fluoride-resistant acid phosphatase. The results show that cholera toxin B subunit–horseradish peroxidase is a more effective retrograde and transganglionic tracer for pelvic primary afferents from the urinary bladder than wheat germ agglutinin–horseradish peroxidase and isolectin B4–horseradish peroxidase, but in contrast to somatic nerves, it is transported mainly by unmyelinated fibres in the visceral afferents.
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early decline and late restoration of spinal cord binding and transganglionic transport of isolectin b4 from Griffonia simplicifolia i after peripheral nerve transection or crush
Restorative Neurology and Neuroscience, 1996Co-Authors: Carl Molander, C Riveromelian, H F Wang, G. GrantAbstract:Copyright (c) 1996 Elsevier Science B.V. All rights reserved. Isolectin B4 from Griffonia simplicifolia I (B4r has a high binding affinity to a large population of unmyelinated primary sensory neurons (Wang et al., Neuroscience 62 (1994r 539−551r. Using immunohistochemical techniques, binding and transganglionic transport of B4 in the spinal cord was investigated, both at short and long survival times, after sciatic nerve transection and ligation or crush in the adult rat. Nerve transection and ligation resulted in nearly complete disappearance of B4 immunolabelling in the sciatic nerve territory of the superficial dorsal horn after B4 binding, as well as after transganglionic transport of B4 by 2 weeks postinjury. Partial recovery of both B4 binding and B4 transport was found by 8 months, and nearly complete recovery by 16 months, indicating that reappearance of B4 binding is not critically dependent on peripheral reinnervation. Crush injury made by jewellers forceps resulted in partial depletion of binding and transport by 2 weeks and a nearly complete recovery by 10 weeks. The results show that binding and transganglionic transport of B4 can be used to label dorsal horn connections of unmyelinated primary afferents during the process of regeneration after crush injury. Furthermore, as B4 binding and transport recover at long survival times in the absence of reestablished peripheral connections, the same techniques can be used to study central primary afferent connections at long survival times after nerve transection. Binding and transganglionic transport of B4 offer alternatives to the use of previous techniques such as transganglionic transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRPr to study central connections of fine primary afferents after injury.
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transganglionic transport and binding of the isolectin b4 from Griffonia simplicifolia i in rat primary sensory neurons
Neuroscience, 1994Co-Authors: H F Wang, Brita Robertson, C Riveromelian, G. GrantAbstract:The isolectin B4 from Griffonia simplicifolia I binds to a subpopulation of rat small-diameter dorsal root ganglion neurons, and to fibres and presumed terminals in laminae I–II of the spinal cord dorsal horn. In the present study we investigated B4 and B4 conjugated to horseradish peroxidase as potential transganglionic tracers of somatic primary afferent neurons after injection into a peripheral nerve. We also tried to identify the specific subpopulation of dorsal root ganglion neurons that bind and transport B4. Following injection of B4 or B4-horseradish peroxidase into the sciatic nerve, labelled presumed terminals that reached peak labelling at two days were found exclusively in regions of the spinal cord gray matter known to receive unmyelinated primary afferent fibres. Almost all dorsal root ganglion cells that transported B4-horseradish peroxidase also bound B4. Cell counts showed that 51% of the dorsal root ganglion neurons were B4-positive and cell area measurements that these were all in the small size range. An extensive overlap was found between B4 and fluoride-resistant acid phosphatase (85%), and between B4 and calcitonin gene-related peptide (59%). Seventeen per cent of the B4-positive cells were substance P-immunoreactive and 9% were immunoreactive to somatostatin. Minimal overlap was seen between B4-positive cells and cells positive for RT97 (3%), a selective marker of primary afferent neurons with myelinated axons. All somatostatin-immunoreactive cells and almost all (95%) of the fluoride-resistant acid phosphatase-positive cells were contained within the B4-positive population. This comprised also 58% of the cells immunoreactive to calcitonin gene-related peptide and 42% of those immunoreactive to substance P. The results obtained show that B4 binds to a subpopulation of unmyelinated primary afferent neurons, and that B4 and B4-horseradish peroxidase can be used as selective transganglionic tracers of this specific cell subpopulation.
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Transganglionic transport and binding of the isolectin B4 from Griffonia simplicifolia I in rat primary sensory neurons.
Neuroscience, 1994Co-Authors: H F Wang, Brita Robertson, C. Rivero-melián, G. GrantAbstract:The isolectin B4 from Griffonia simplicifolia I binds to a subpopulation of rat small-diameter dorsal root ganglion neurons, and to fibres and presumed terminals in laminae I-II of the spinal cord dorsal horn. In the present study we investigated B4 and B4 conjugated to horseradish peroxidase as potential transganglionic tracers of somatic primary afferent neurons after injection into a peripheral nerve. We also tried to identify the specific subpopulation of dorsal root ganglion neurons that bind and ganglion neurons that bind and transport B4. Following injection of B4 or B4-horseradish peroxidase into the sciatic nerve, labelled presumed terminals that reached peak labelling at two days were found exclusively in regions of the spinal cord gray matter known to receive unmyelinated primary afferent fibres. Almost all dorsal root ganglion cells that transported B4-horseradish peroxidase also bound B4. Cell counts showed that 51% of the dorsal root ganglion neurons were B4-positive and cell area measurements that these were all in the small size range. An extensive overlap was found between B4 and fluoride-resistant acid phosphatase (85%), and between B4 and calcitonin gene-related peptide (59%). Seventeen per cent of the B4-positive cells were substance P-immunoreactive and 9% were immunoreactive to somatostatin. Minimal overlap was seen between B4-positive cells and cells positive for RT97 (3%), a selective marker of primary afferent neurons with myelinated axons. All somatostatin-immunoreactive cells and almost all (95%) of the fluoride-resistant acid phosphatase-positive cells were contained within the B4-positive population. This comprised also 58% of the cells immunoreactive to calcitonin gene-related peptide and 42% of those immunoreactive to substance P. The results obtained show that B4 binds to a subpopulation of unmyelinated primary afferent neurons, and that B4 and B4-horseradish peroxidase can be used as selective transganglionic tracers of this specific cell subpopulation.
Irwin J. Goldstein - One of the best experts on this subject based on the ideXlab platform.
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comparison of the binding properties of the mushroom marasmius oreades lectin and Griffonia simplicifolia i b4 isolectin to αgalactosyl carbohydrate antigens in the surface phase
Xenotransplantation, 2004Co-Authors: Svend Kirkeby, Harry C Winter, Irwin J. GoldsteinAbstract:Kirkeby S, Winter HC, Goldstein IJ. Comparison of the binding properties of the mushroom Marasmius oreades lectin and Griffonia simplicifolia I-B4 isolectin to agalactosyl carbohydrate antigens in the surface phase. Xenotransplantation 2004; 11: 254-261. � Blackwell Munksgaard, 2004 Abstract: The binding of two a-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B4 (GS I-B4 )t o neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various a-galactosylated glycans and laminin from basement membrane of mouse sarcoma that contains the xenogenic Gala1-3Gal1-4GlcNAc epitope were immobilized in microtiter plate wells and lectin binding determined with an enzyme- linked assay. After 24 h of incubation, MOA had higher affinity for the xenogenic pentasaccharide (Gala1-3Gal1-4GlcNAcb1-3Galb1-4Glc) than for the Gala-monosaccharide. The binding properties of MOA and GS I-B4 to the xenogenic disaccharide (Gala1-3Galb1) were comparable while the binding of MOA to the xenogenic pentasaccharide was much stronger than the binding of GS I-B4 to the same epitope. Non-xeno- genic disaccharide-coupled neoglycoproteins having galactose end groups linked a1-2 or a1-4 to Gal or linked a1-3 to GalNAc bound very weakly to MOA, whereas GS I-B4 recognized all of these disac- charides with similarly high affinity. MOA also showed high affinity for laminin. The results indicate that the Marasmius oreades lectin has nearly the same affinities as does GS I-B4 for the simple xenogenic carbohydrate antigens, but MOA has greater affinity for the pentasac- charide and is far more specific in its binding preferences than the Griffonia lectin.
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comparison of the binding properties of the mushroom marasmius oreades lectin and Griffonia simplicifolia i b4 isolectin to αgalactosyl carbohydrate antigens in the surface phase
Xenotransplantation, 2004Co-Authors: Svend Kirkeby, Harry C Winter, Irwin J. GoldsteinAbstract:The binding of two alpha-galactophilic lectins, Marasmius oreades agglutinin (MOA), and Griffonia simplicifolia I isolectin B(4) (GS I-B(4)) to neoglycoproteins and natural glycoproteins were compared in a surface phase assay. Neoglycoproteins carrying various alpha-galactosylated glycans and laminin from basement membrane of mouse sarcoma that contains the xenogenic Galalpha1-3Gal1-4GlcNAc epitope were immobilized in microtiter plate wells and lectin binding determined with an enzyme-linked assay. After 24 h of incubation, MOA had higher affinity for the xenogenic pentasaccharide (Galalpha1-3Gal1-4GlcNAcbeta1-3Galbeta1-4Glc) than for the Galalpha-monosaccharide. The binding properties of MOA and GS I-B(4) to the xenogenic disaccharide (Galalpha1-3Galbeta1) were comparable while the binding of MOA to the xenogenic pentasaccharide was much stronger than the binding of GS I-B(4) to the same epitope. Non-xenogenic disaccharide-coupled neoglycoproteins having galactose end groups linked alpha1-2 or alpha1-4 to Gal or linked alpha1-3 to GalNAc bound very weakly to MOA, whereas GS I-B(4) recognized all of these disaccharides with similarly high affinity. MOA also showed high affinity for laminin. The results indicate that the Marasmius oreades lectin has nearly the same affinities as does GS I-B(4) for the simple xenogenic carbohydrate antigens, but MOA has greater affinity for the pentasaccharide and is far more specific in its binding preferences than the Griffonia lectin.
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Facile preparation of the α-Gal-recognizing Griffonia simplicifolia I-B4 isolectin
Carbohydrate research, 2004Co-Authors: Harry C Winter, Irwin J. GoldsteinAbstract:The B4 isolectin from Griffonia simplicifolia is of great utility as a reagent for the identification of alpha-D-galactopyranosyl end groups. Its separation from isolectins containing A subunits has been greatly improved by a simple, rapid procedure using a column of N-acetylgalactosamine coupled to vinyl sulfone-activated Sepharose 4B to selectively retain the A subunit-containing isolectins. The procedure has the advantages over previous affinity procedures of speed (the isolation of B4 isolectin can be achieved in one day), simplicity, and high degree of resolution of the B4 isolectin.
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The role of valence on the high-affinity binding of Griffonia simplicifolia isolectins to type A human erythrocytes
Biochemistry, 1998Co-Authors: Randall N. Knibbs, Masaru Takagaki, Diane A. Blake, Irwin J. GoldsteinAbstract:The Griffonia simplicifolia-I (GS-I) isolectins have been used to probe the effect of lectin valence on their high-affinity binding to human erythrocytes. These tetrameric lectins are composed of A and B subunits and constitute a series of five isolectins (A4, A3B, A2B2, AB3, B4). The A subunit is specific for alpha-D-GalNAc end groups and binds to the blood type A determinant GalNAcalpha1, as well as to terminal alpha-D-Gal groups found on type B cells. The B subunit is specific for alpha-D-Gal end groups, and binds very specifically to type B erythrocytes. This series of isolectins is tetravalent (A4), trivalent (A3B), divalent (A2B2), and monovalent (AB3) for type A erythrocytes; thus, this system provides the opportunity to examine the effect of lectin valency on the association constants of these GS-I isolectins binding to cells. Cell binding experiments carried out using 125I-labeled GS-I isolectins and type A human erythrocytes allowed us to demonstrate that (1) the association constant of the isolectin monovalent for alpha-D-GalNAc (AB3) is virtually identical to its association constant for the haptenic sugar methyl-N-acetyl-alpha-D-galactosaminide, reported previously, and (2) the association constant of the GS-I isolectins for human type A erythrocytes increases with increasing valency of the isolectin. These results indicate that the increased affinity displayed by the GS-I isolectins for human type A erythrocytes is dependent on their multivalency, and not on an extended binding site nor on nonspecific, or noncarbohydrate, interactions of the lectin with the cell surface. These findings should be of general relevance to understanding the high-affinity interactions observed between other multivalent proteins and multivalent ligands (e.g., cell surfaces).
H F Wang - One of the best experts on this subject based on the ideXlab platform.
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retrograde and transganglionic transport of horseradish peroxidase conjugated cholera toxin b subunit wheatgerm agglutinin and isolectin b4 from Griffonia simplicifolia i in primary afferent neurons innervating the rat urinary bladder
Neuroscience, 1998Co-Authors: H F Wang, Peter Shortland, M J Park, G. GrantAbstract:In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6–S1 spinal cord segments and in the T13–L2 and L6–S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit–horseradish peroxidase was injected. Cholera toxin B subunit–horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV–VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin–horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit–horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I–horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit–horseradish peroxidase than with wheat germ agglutinin– or isolectin B4–horseradish peroxidase. In the L6–S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with myelinated fibres). Double labelling with other neuronal markers showed that 71%, 43% and 36% of the cholera toxin B subunit-immunoreactive cells were calcitonin gene-related peptide-, isolectin B4-binding- and substance P-positive, respectively. A few cholera toxin B subunit cells showed galanin-immunoreactivity, but none were somatostatin-, vasoactive intestinal polypeptide-, or neuropeptide Y-immunoreactive or contained fluoride-resistant acid phosphatase. The results show that cholera toxin B subunit–horseradish peroxidase is a more effective retrograde and transganglionic tracer for pelvic primary afferents from the urinary bladder than wheat germ agglutinin–horseradish peroxidase and isolectin B4–horseradish peroxidase, but in contrast to somatic nerves, it is transported mainly by unmyelinated fibres in the visceral afferents.
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early decline and late restoration of spinal cord binding and transganglionic transport of isolectin b4 from Griffonia simplicifolia i after peripheral nerve transection or crush
Restorative Neurology and Neuroscience, 1996Co-Authors: Carl Molander, C Riveromelian, H F Wang, G. GrantAbstract:Copyright (c) 1996 Elsevier Science B.V. All rights reserved. Isolectin B4 from Griffonia simplicifolia I (B4r has a high binding affinity to a large population of unmyelinated primary sensory neurons (Wang et al., Neuroscience 62 (1994r 539−551r. Using immunohistochemical techniques, binding and transganglionic transport of B4 in the spinal cord was investigated, both at short and long survival times, after sciatic nerve transection and ligation or crush in the adult rat. Nerve transection and ligation resulted in nearly complete disappearance of B4 immunolabelling in the sciatic nerve territory of the superficial dorsal horn after B4 binding, as well as after transganglionic transport of B4 by 2 weeks postinjury. Partial recovery of both B4 binding and B4 transport was found by 8 months, and nearly complete recovery by 16 months, indicating that reappearance of B4 binding is not critically dependent on peripheral reinnervation. Crush injury made by jewellers forceps resulted in partial depletion of binding and transport by 2 weeks and a nearly complete recovery by 10 weeks. The results show that binding and transganglionic transport of B4 can be used to label dorsal horn connections of unmyelinated primary afferents during the process of regeneration after crush injury. Furthermore, as B4 binding and transport recover at long survival times in the absence of reestablished peripheral connections, the same techniques can be used to study central primary afferent connections at long survival times after nerve transection. Binding and transganglionic transport of B4 offer alternatives to the use of previous techniques such as transganglionic transport of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRPr to study central connections of fine primary afferents after injury.
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transganglionic transport and binding of the isolectin b4 from Griffonia simplicifolia i in rat primary sensory neurons
Neuroscience, 1994Co-Authors: H F Wang, Brita Robertson, C Riveromelian, G. GrantAbstract:The isolectin B4 from Griffonia simplicifolia I binds to a subpopulation of rat small-diameter dorsal root ganglion neurons, and to fibres and presumed terminals in laminae I–II of the spinal cord dorsal horn. In the present study we investigated B4 and B4 conjugated to horseradish peroxidase as potential transganglionic tracers of somatic primary afferent neurons after injection into a peripheral nerve. We also tried to identify the specific subpopulation of dorsal root ganglion neurons that bind and transport B4. Following injection of B4 or B4-horseradish peroxidase into the sciatic nerve, labelled presumed terminals that reached peak labelling at two days were found exclusively in regions of the spinal cord gray matter known to receive unmyelinated primary afferent fibres. Almost all dorsal root ganglion cells that transported B4-horseradish peroxidase also bound B4. Cell counts showed that 51% of the dorsal root ganglion neurons were B4-positive and cell area measurements that these were all in the small size range. An extensive overlap was found between B4 and fluoride-resistant acid phosphatase (85%), and between B4 and calcitonin gene-related peptide (59%). Seventeen per cent of the B4-positive cells were substance P-immunoreactive and 9% were immunoreactive to somatostatin. Minimal overlap was seen between B4-positive cells and cells positive for RT97 (3%), a selective marker of primary afferent neurons with myelinated axons. All somatostatin-immunoreactive cells and almost all (95%) of the fluoride-resistant acid phosphatase-positive cells were contained within the B4-positive population. This comprised also 58% of the cells immunoreactive to calcitonin gene-related peptide and 42% of those immunoreactive to substance P. The results obtained show that B4 binds to a subpopulation of unmyelinated primary afferent neurons, and that B4 and B4-horseradish peroxidase can be used as selective transganglionic tracers of this specific cell subpopulation.
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Transganglionic transport and binding of the isolectin B4 from Griffonia simplicifolia I in rat primary sensory neurons.
Neuroscience, 1994Co-Authors: H F Wang, Brita Robertson, C. Rivero-melián, G. GrantAbstract:The isolectin B4 from Griffonia simplicifolia I binds to a subpopulation of rat small-diameter dorsal root ganglion neurons, and to fibres and presumed terminals in laminae I-II of the spinal cord dorsal horn. In the present study we investigated B4 and B4 conjugated to horseradish peroxidase as potential transganglionic tracers of somatic primary afferent neurons after injection into a peripheral nerve. We also tried to identify the specific subpopulation of dorsal root ganglion neurons that bind and ganglion neurons that bind and transport B4. Following injection of B4 or B4-horseradish peroxidase into the sciatic nerve, labelled presumed terminals that reached peak labelling at two days were found exclusively in regions of the spinal cord gray matter known to receive unmyelinated primary afferent fibres. Almost all dorsal root ganglion cells that transported B4-horseradish peroxidase also bound B4. Cell counts showed that 51% of the dorsal root ganglion neurons were B4-positive and cell area measurements that these were all in the small size range. An extensive overlap was found between B4 and fluoride-resistant acid phosphatase (85%), and between B4 and calcitonin gene-related peptide (59%). Seventeen per cent of the B4-positive cells were substance P-immunoreactive and 9% were immunoreactive to somatostatin. Minimal overlap was seen between B4-positive cells and cells positive for RT97 (3%), a selective marker of primary afferent neurons with myelinated axons. All somatostatin-immunoreactive cells and almost all (95%) of the fluoride-resistant acid phosphatase-positive cells were contained within the B4-positive population. This comprised also 58% of the cells immunoreactive to calcitonin gene-related peptide and 42% of those immunoreactive to substance P. The results obtained show that B4 binds to a subpopulation of unmyelinated primary afferent neurons, and that B4 and B4-horseradish peroxidase can be used as selective transganglionic tracers of this specific cell subpopulation.
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Calcium modulates protease resistance and carbohydrate binding of a plant defense legume lectin, Griffonia simplicifolia lectin II (GSII).
Comparative Biochemistry and Physiology B, 2002Co-Authors: Keyan Zhu-salzman, Ron A Salzman, Hisashi Koiwa, Philip K. Hammen, Ray A. Bressan, Larry L. Murdock, Paul M. HasegawaAbstract:Site-directed mutagenesis previously identified the residues responsible for the biological activity of the plant defense legume lectin, Griffonia simplicifolia lectin II (GSII) wProc. Natl. Acad. Sci. USA 95, (1998) 15123–15128x. However, these results were inconclusive as to whether these residues function as direct defense determinants through carbohydrate binding, or whether substantial changes of the protein structure had occurred in mutated proteins, with this structural disruption actually causing the loss of biochemical and biological functions. Evidence shown here supports the former explanation: circular dichroism and fluorescence spectra showed that mutations at carbohydrate-binding residues of GSII do not render it disfunctional because of substantial secondary or tertiary structure modifications; and trypsin treatment confirmed that rGSII structural integrity is retained in these mutants. Reduced biochemical stability was observed through papain digestion and urea denaturation in mutant versions that had lost carbohydrate-binding ability, and this was correlated with lower Ca content. Accordingly, the re-addition of Ca to demetalized proteins could recover resistance 2q 2q
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Carbohydrate binding and resistance to proteolysis control insecticidal activity of Griffonia simplicifolia lectin II
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Keyan Zhu-salzman, Ron A Salzman, Hisashi Koiwa, Richard E Shade, Ray A. Bressan, Paul M. Hasegawa, Meena L. Narasimhan, Larry L. MurdockAbstract:Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.
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identification of n acetylglucosamine binding residues in Griffonia simplicifolia lectin ii
FEBS Letters, 1996Co-Authors: Keyan Zhu, Ray A. Bressan, Paul M. Hasegawa, Larry L. MurdockAbstract:Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp88, Glu108, Tyr134, Asn136, Leu226 and Gln227) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp88, Tyr134 or Asn136 eliminated GlcNAc binding activity by GS-II, while those at Glut108, Leu226 or Gln227 did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr134 eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu226 and Gln227 did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.
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An Insecticidal N-Acetylglucosamine-Specific Lectin Gene from Griffonia simplicifolia (Leguminosae)
Plant Physiology, 1996Co-Authors: Joseph E. Huesing, Richard E Shade, Ray A. Bressan, Paul M. Hasegawa, Larry L. MurdockAbstract:Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.
