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Peter Bohlen - One of the best experts on this subject based on the ideXlab platform.

  • Refolding and characterization of human recombinant heparin-binding Neurite-Promoting Factor.
    Protein expression and purification, 1994
    Co-Authors: Andrew P. Seddon, Imre Kovesdi, J.d. Hulmes, Mildred Decker, Jeanette L. Fairhurst, Joseph M. Backer, M. Doughervermazen, Peter Bohlen
    Abstract:

    Abstract Heparin-binding Neurite-Promoting Factor (HBNF) is a highly basic, cysteine-rich 136-residue protein, and a member of a new class of heparin-binding proteins. It exhibits a Neurite-outgrowth Promoting activity and its expression is both temporally and spacially regulated during fetal and postnatal development. A high inter-species sequence conservation suggests important, presently unknown, biological functions. HBNF is structurally and most likely functionally related to the product of a developmentally regulated gene, MK (midkine). To elucidate biological roles of these proteins, recombinant forms of the proteins were produced. Expression of human recombinant HBNF and MK in Escherichia coli lead to the formation of insoluble aggregated protein that accounted for about 25% of the total cellular protein. Homogeneous, monomeric forms of each protein were recovered from inclusion bodies by reduction with dithiothreitol and solubilization in 8 M urea. Re-folding of the reduced and denatured protein occurred upon dialysis at pH 7.4. Human recombinant (hr) HBNF and hrMK prepared in this manner were further purified by heparin affinity chromatography. Chromatographic evidence demonstrates that refolding and concomitant disulfide bond formation in hrHBNF proceeds in high yield with minimal formation of stable nonnative disulfides. Studies on the redox status of the 10 cysteine residues of bovine brain HBNF and the refolded recombinant protein indicate that all cysteines are engaged in disulfide bond formation. The disulfide arrangements for the recombinant protein were found to be identical to those in the native protein isolated from bovine brain. We also found that correctly folded HBNF and MK inhibit the binding of FGF-2 to its high affinity receptor. Thus, biologic and biochemical evidence suggest that recombinant and tissue-derived HBNF are structurally and functionally equivalent and that function is dependent on proper folding of the proteins.

  • Comparison of the disulfide bond arrangements of human recombinant and bovine brain heparin binding Neurite-Promoting Factors.
    Biochemical and biophysical research communications, 1993
    Co-Authors: J.d. Hulmes, Andrew P. Seddon, Mildred Decker, Peter Bohlen
    Abstract:

    Heparin binding Neurite-Promoting Factor (HBNF) is a highly basic 136 amino acid protein containing 10 cysteine residues. We have determined the redox status and the disulfide arrangement of the cysteine residues in HBNF from bovine brain and refolded human recombinant protein produced in E. coli. Our data indicate that all 10 cysteines are involved in disulfide bond formation. The disulfide linkages of human recombinant and bovine brain HBNF, as determined after proteolytic digestions of the non-reduced proteins by peptide mapping and sequence analysis are: Cys15-Cys44, Cys23-Cys53, Cys30-Cys57, Cys67-Cys99 and Cys77-Cys109. Thus, recombinant HBNF has the same disulfide arrangement as the native brain-derived protein.

  • Expression of the HBNF (heparin-binding Neurite-Promoting Factor) gene in the brain of fetal, neonatal and adult rat: an in situ hybridization study.
    Brain research. Developmental brain research, 1992
    Co-Authors: Bertrand Bloch, Imre Kovesdi, Elisabeth Normand, Peter Bohlen
    Abstract:

    Abstract HBNF (heparin-binding Neurite-Promoting Factor) and MK (midkine) are members of a newly recognized family of proteins, the expression of which is developmentally regulated. These proteins are expressed highest during fetal development in many tissues but they seem to be rather restricted to the brain in adult animals. Gene expression for these proteins is inducible by retinoic acid in embryonal carcinoma cell lines. They induce Neurite outgrowth in cultured neurons, and they are characterized by high sequence conservation between species. While the function(s) of these proteins are unknown, available evidence suggests possible roles in the development and the maintenance of neural tissues. This in situ hybridization study investigates the temporal and spatial expression pattern of the HBNF gene in the brain of developing rats. The HBNF gene is highly expressed in the neuroepithelium and the ependyma from fetal day 15 on. Although most ependymal structures express the gene strongly, a few restricted areas of the ependyma do not express HBNF (ventral part of the fourth ventricle, subcommissural organ). In the brain parenchyma, HBNF is expressed in the thalamo-hippocampal area from fetal day 15 and in the cerebral cortex from fetal day 16, with high expression occuring in the superficial layers of the cortex. The nature of the cells expressing the gene, while difficult to ascertain, is probably glial for the most part. However, certain neurons (in limited areas of the brain parenchyma) and most pial cells (in the meninges), also express the gene, HBNF gene expression decreases sharply a few days after birth. HBNF mRNA is also detectable at fetal days 15 and 16 in the face fetal mesenchyma. In the adult rat brain, the expression of the HBNF gene appears to be restricted to neurons of the hippocampus and of the olFactory bulb and to the superficial layers of the cortex. The structurally related MK gene, though not extensively studied here, shows an entirely different temporal and spatial expression pattern. MK gene is weakly expressed during ontogeny in most brain areas, and in the adult animal, MK mRNA is present only in the choroid plexus. The intense and widely distributed expression of the HBNF gene in several cell populations in the fetus, the progressive spatial and quantitative restriction of HBNF gene expression with brain differentiation, as well as the properties of the protein suggest important and diverse functions for HBNF in cellular interactions and cell differentiation in the developing brain, that must act temporally and spatially by ways distinct from its MK companion molecule.

  • HBNF and MK, members of a novel gene family of heparin-binding proteins with potential roles in embryogenesis and brain function
    Progress in Growth Factor Research, 1991
    Co-Authors: Peter Bohlen, Imre Kovesdi
    Abstract:

    HBNF (heparin-binding Neurite-Promoting Factor) is a heparin-binding protein which is found primarily in the brain and stimulates Neurite outgrowth in cultured perinatal neurons. It was also reported to be mitogenic for fibroblasts and endothelial cells but this activity is still controversial. The sequence of HBNF is highly conserved in diverse species suggesting important function. Expression of the HBNF gene in brain tissue appears to be developmentally regulated, increasing during gestation to highest levels around the time of birth. The HBNF gene shows high sequence homology to another gene, MK (midkine). Like HBNF, the MK gene is developmentally regulated, however, high expression occurs in most fetal tissues during mid-gestation. The biological properties of the MK protein are remarkably similar to those of HBNF. The available evidence suggests that HBNF and MK are members of a new family of genes with potential roles in fetal development and in brain function or maintenance.

Masaki Nodal - One of the best experts on this subject based on the ideXlab platform.

  • 1α,25-Dihydroxyvitamin D_3 down-regulates pleiotrophin messenger RNA expression in osteoblast-like cells
    Endocrine, 1995
    Co-Authors: Masato Tamura, Fumihiko Ichikawa, R. Paul Guillerman, Thomas F. Deuel, Masaki Nodal
    Abstract:

    Pleiotrophin (PTN)[heparin-binding-growth-associated molecule (HB-GAM), heparin-binding Neurite-Promoting Factor (HBNF)] is a recently identified polypeptide that stimulates growth of fibroblasts and enhances Neurite extension. PTN is expressed in many tissues but relatively high level of expression has been observed in brain and bone. We examined hormonal regulation of PTN mRNA expression in several osteoblast-like cell lines including MC3T3-E1 and ROS17/2.8. The levels of PTN mRNA in these cells was significantly reduced by treatment with 10^−8 m 1α,25-dihydroxyvitamin D_3 (1,25(OH)_2D_3) for 24 h. However, PTN mRNA levels were increased when the non-osteoblastic cell line, ROS 25/1, was treated with 1,25(OH)_2D_3. These effects were observed in a dose-dependent manner in a dose range between 10^−11 m to 10^−8 m . This effect was specific to 1,25(OH)_2D_3, since PTN mRNA levels were not affected by other steroids such as retinoic acid and dexamethasone in MC3T3-E1 or ROS17/2.8 cells. Similar 1,25(OH)_2D_3 down-regulation of PTN mRNA was also observed in primary cultures of osteoblast-enriched fetal rat calvaria cells as well as cultures of MC3T3-E1 and ROS17/2.8 cells. These observations suggest that PTN expression in osteoblasts is regulated by the calcitropic hormone, 1,25(OH)_2D_3, and that PTN may play a role in vitamin D-dependent regulation of bone metabolism.

  • 1α,25-Dihydroxyvitamin D3 down-regulates pleiotrophin messenger RNA expression in osteoblast-like cells
    Endocrine, 1995
    Co-Authors: Masato Tamura, Fumihiko Ichikawa, R. Paul Guillerman, Thomas F. Deuel, Masaki Nodal
    Abstract:

    Pleiotrophin (PTN)[heparin-binding-growth-associated molecule (HB-GAM), heparin-binding Neurite-Promoting Factor (HBNF)] is a recently identified polypeptide that stimulates growth of fibroblasts and enhances Neurite extension. PTN is expressed in many tissues but relatively high level of expression has been observed in brain and bone. We examined hormonal regulation of PTN mRNA expression in several osteoblast-like cell lines including MC3T3-E1 and ROS17/2.8. The levels of PTN mRNA in these cells was significantly reduced by treatment with 10−8 m 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 24 h. However, PTN mRNA levels were increased when the non-osteoblastic cell line, ROS 25/1, was treated with 1,25(OH)2D3. These effects were observed in a dose-dependent manner in a dose range between 10−11 m to 10−8 m. This effect was specific to 1,25(OH)2D3, since PTN mRNA levels were not affected by other steroids such as retinoic acid and dexamethasone in MC3T3-E1 or ROS17/2.8 cells. Similar 1,25(OH)2D3 down-regulation of PTN mRNA was also observed in primary cultures of osteoblast-enriched fetal rat calvaria cells as well as cultures of MC3T3-E1 and ROS17/2.8 cells. These observations suggest that PTN expression in osteoblasts is regulated by the calcitropic hormone, 1,25(OH)2D3, and that PTN may play a role in vitamin D-dependent regulation of bone metabolism.

Mats Paulsson - One of the best experts on this subject based on the ideXlab platform.

  • Merosin promotes cell attachment and Neurite outgrowth and is a component of the Neurite-Promoting Factor of RN22 schwannoma cells.
    Experimental cell research, 1992
    Co-Authors: Eva Engvall, Marston Manthorpe, Diane Earwicker, Adrienne Day, David Muir, Mats Paulsson
    Abstract:

    The laminin-like protein merosin was purified from human placenta in intact form and as pepsin fragments and compared to laminin in heparin affinity chromatography and cell binding assays. Intact merosin and a small fragment of merosin comprising the last two repeats of the heavy chain g domain bind to heparin. Intact merosin and large pepsin fragments of merosin, but not the small C-terminal fragment, mediate the attachment and spreading of several types of cells and promote Neurite outgrowth from neuronal cells similar to laminin and its corresponding fragments. Cells with various integrin-type receptors for laminin attached equally well to merosin and laminin, suggesting that several of the known laminin binding receptors also bind to merosin. Antibodies to the beta 1 subunit of integrins inhibited Neurite outgrowth on merosin as well as on laminin, confirming the involvement of integrin-mediated interaction of cells with both merosin and laminin. Schwannoma cells, which have previously been shown to produce a laminin-like, Neurite-Promoting Factor, synthesize merosin in vivo and in vitro as shown by protein and mRNA analysis. The results suggest that merosin, which is the more abundant basement membrane protein in the laminin family, has properties very similar to laminin despite differences in the structure of the heavy chain. Furthermore, merosin may be identical to or a component of the Neurite-Promoting Factors previously reported from heart, muscle, and Schwann cells.

Ronald P. Hart - One of the best experts on this subject based on the ideXlab platform.

  • Gene expression profiling of acute spinal cord injury reveals spreading inflammatory signals and neuron loss
    Physiological genomics, 2001
    Co-Authors: Jason B. Carmel, Anthony Galante, Patricia Soteropoulos, Peter Tolias, Michael Recce, Wise Young, Ronald P. Hart
    Abstract:

    We have completed the first large-scale gene expression study of acute spinal cord injury (SCI) in rat. Oligonucleotide microarrays containing 1,200 gene-specific probes were used to quantify mRNA levels, relative to uninjured controls, in spinal cords injured using a standard contusion model. Our results revealed a marked loss of neuron-specific mRNAs at the injury site. The surviving cells showed a characteristic inflammatory response that started at the injury site and spread to the distal cord. Changes in several mRNA levels were associated with putative regenerative responses in the spinal cord. Notably, phosphodiesterase 4, nestin, glia-derived Neurite Promoting Factor, and GAP-43 mRNAs increased significantly. Other mRNAs clustered temporally and spatially with these regeneration-associated genes. Thus we have described global patterns of gene expression following acute SCI, and we have identified targets for future study and possible therapeutic intervention.

Masato Tamura - One of the best experts on this subject based on the ideXlab platform.

  • 1α,25-Dihydroxyvitamin D_3 down-regulates pleiotrophin messenger RNA expression in osteoblast-like cells
    Endocrine, 1995
    Co-Authors: Masato Tamura, Fumihiko Ichikawa, R. Paul Guillerman, Thomas F. Deuel, Masaki Nodal
    Abstract:

    Pleiotrophin (PTN)[heparin-binding-growth-associated molecule (HB-GAM), heparin-binding Neurite-Promoting Factor (HBNF)] is a recently identified polypeptide that stimulates growth of fibroblasts and enhances Neurite extension. PTN is expressed in many tissues but relatively high level of expression has been observed in brain and bone. We examined hormonal regulation of PTN mRNA expression in several osteoblast-like cell lines including MC3T3-E1 and ROS17/2.8. The levels of PTN mRNA in these cells was significantly reduced by treatment with 10^−8 m 1α,25-dihydroxyvitamin D_3 (1,25(OH)_2D_3) for 24 h. However, PTN mRNA levels were increased when the non-osteoblastic cell line, ROS 25/1, was treated with 1,25(OH)_2D_3. These effects were observed in a dose-dependent manner in a dose range between 10^−11 m to 10^−8 m . This effect was specific to 1,25(OH)_2D_3, since PTN mRNA levels were not affected by other steroids such as retinoic acid and dexamethasone in MC3T3-E1 or ROS17/2.8 cells. Similar 1,25(OH)_2D_3 down-regulation of PTN mRNA was also observed in primary cultures of osteoblast-enriched fetal rat calvaria cells as well as cultures of MC3T3-E1 and ROS17/2.8 cells. These observations suggest that PTN expression in osteoblasts is regulated by the calcitropic hormone, 1,25(OH)_2D_3, and that PTN may play a role in vitamin D-dependent regulation of bone metabolism.

  • 1α,25-Dihydroxyvitamin D3 down-regulates pleiotrophin messenger RNA expression in osteoblast-like cells
    Endocrine, 1995
    Co-Authors: Masato Tamura, Fumihiko Ichikawa, R. Paul Guillerman, Thomas F. Deuel, Masaki Nodal
    Abstract:

    Pleiotrophin (PTN)[heparin-binding-growth-associated molecule (HB-GAM), heparin-binding Neurite-Promoting Factor (HBNF)] is a recently identified polypeptide that stimulates growth of fibroblasts and enhances Neurite extension. PTN is expressed in many tissues but relatively high level of expression has been observed in brain and bone. We examined hormonal regulation of PTN mRNA expression in several osteoblast-like cell lines including MC3T3-E1 and ROS17/2.8. The levels of PTN mRNA in these cells was significantly reduced by treatment with 10−8 m 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 24 h. However, PTN mRNA levels were increased when the non-osteoblastic cell line, ROS 25/1, was treated with 1,25(OH)2D3. These effects were observed in a dose-dependent manner in a dose range between 10−11 m to 10−8 m. This effect was specific to 1,25(OH)2D3, since PTN mRNA levels were not affected by other steroids such as retinoic acid and dexamethasone in MC3T3-E1 or ROS17/2.8 cells. Similar 1,25(OH)2D3 down-regulation of PTN mRNA was also observed in primary cultures of osteoblast-enriched fetal rat calvaria cells as well as cultures of MC3T3-E1 and ROS17/2.8 cells. These observations suggest that PTN expression in osteoblasts is regulated by the calcitropic hormone, 1,25(OH)2D3, and that PTN may play a role in vitamin D-dependent regulation of bone metabolism.