gelatin-Sepharose 4B Research Articles

Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro

Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts.

The Rough Endoplasmic Reticulum-resident FK506-binding Protein FKBP65 Is a Molecular Chaperone That Interacts with Collagens

The rough endoplasmic reticulum-resident FK-506-binding protein FKBP65 can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. The peptidylprolyl cis-trans-isomerase activity of FKBP65 was previously shown to have only marginal effects on the rate of triple helix formation (Zeng, B., MacDonald, J. R., Bann, J. G., Beck, K., Gambee, J. E., Boswell, B. A., and Bächinger, H. P. (1998) Biochem. J. 330, 109-114). Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.

フィブロネクチン点眼剤調製の自動化への試み

To develop an automatic device for the preparation of fibronectin eyedrops a basic study was performed to establish the optimum conditions for the preparation procedures. Fibronectin eyedrops were prepared according to the method of Nishida et al. In the first step with gelatinsepharose 4B (GS4B), the optimum conditions were as follows:(1) At least five and 15 ml of phosphate buffered saline (PBS) containing citrate were needed to wash the GS4B before and after plasma treatment, respectively;(2) The economical optimum ratio of GS4B vs. human plsama was 1: 10;(3) Preparations at either low or high temperatures should be avoided (optimum temperature 22°C); and (4) Higher yields of fibronectin were obtained with slower plasma flow rates. In the second step with sephadex-G25 (PD-10):(1) 20 ml of PBS were needed for pre-washing of PD-10; and (2) A high concentration and high yield fraction of fibronectin could be obtained when the eluate of 4 M urea from GS4B containing fibronectin was directly infused into PD-10. These results are considered important for the effective preparation of fibronectin eyedrops either automatically or manually.

Zymographic measurement of gelatinase activity in brain tissue after detergent extraction and affinity-support purification

Several methods have been developed for the measurement of gelatinase activity from various tissues using detergent extraction. Gelatin-affinity chromatography has been employed for the large-scale purification of gelatinases from conditioned medium obtained from cultured cells. The objective of this paper was to develop a rapid method whereby gelatinase activity could be extracted from regional brain tissues without tedious, intervening purification steps. After Triton X-100 extraction and gelatin-Sepharose 4B purification of rat brain tissue extracts, two major activities were observed on gelatin zymograms. These were identified as gelatinase A and B using co-migration with astrocyte-derived enzymes and inhibition of activity by tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). The non-ionic detergents, Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) were equally effective in extracting activities from brain tissue. Little difference in recovery was observed among 0.1, 1 and 10% concentrations of Triton X-100. The method developed here was capable of recovering gelatinase activities from rat brain tissue over a 4–10-fold range using gelatin zymography for the measurement of activity. It is possible that this method may be modified for the measurement of gelatinases in tissues such as biopsy samples of gliomas or astrocytomas or other cancers where gelatinases are thought to play a role in tumor invasion and/or metastasis.

Role of the matrixin MMP-2 in multicellular organization of adipocytes cultured in basement membrane components.

Primary rat adipocytes cultured in basement membrane component gels migrated and organized into large, three-dimensional, multicellular clusters. Gross morphological changes seen during this reorganization are described. The rate of cluster formation decreased with age of the rats and was stimulated by insulin in older, but not in younger rats. Echistatin, a disintegrin, partially inhibited the formation of multicellular clusters in a concentration-dependent fashion (50% inhibitory concentration approximately 10 nM). The original extracellular matrix was initially remodeled and eventually destroyed by the time large multicellular clusters were observed. This implied that one or more matrix-degrading protease(s) were being secreted. Adipocyte-conditioned medium was found to contain a divalent cation-sensitive gelatinase activity at approximately 72 and/or approximately 62 kDa. The elution profile of this activity from gelatin-Sepharose 4B was similar to matrix metalloproteinase 2 (MMP-2, a 72-kDa matrixin with a 62-kDa mature form), and the dimethyl sulfoxide eluant from these columns contained MMP-2 immunoreactivity. MMP-2 concentration and activity were greater in conditioned medium from young than from older animals; however, insulin did not affect the amount of MMP-2 in adipocyte-conditioned media. The matrixin inhibitor 1,10-phenanthroline not only blocked gelatinase activity in zymograms but also prevented extracellular matrix remodeling and destruction, as well as adipocyte migration and the formation of cell-cell contacts in adipocyte cultures. These observations are consistent with the hypothesis that the matrixin MMP-2 is secreted by adipocytes. Whereas matrixin activity alone may not be sufficient for the formation of multicellular clusters, the data indicate that it may have a requisite role in this process.

Endoplasmic reticulum protein Hsp47 binds specifically to the N-terminal globular domain of the amino-propeptide of the procollagen I alpha 1 (I)-chain.

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts, has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with 35[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro alpha 1(I)-N-propeptide at 25 micrograms/ml completely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro alpha 1(I)-N-propeptide domain were confirmed by examination of polysome-associated pro alpha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1 (I)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23-108 (NP1), 23-151 (NP2), and 23-178 (NP3) within the pro alpha 1 (I)- N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro alpha 1(I)- and pro alpha 2(I)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha 2(I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a "chaperone" role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the pro alpha 1 (I) chain, and the pro alpha 1(I) amino-propeptide region in particular.

Purification of human neutrophil collagenase, establishment of its monoclonal antibodies and application to gingival crevicular neutrophils

In order to investigate the role of neutrophil collagenase in the periodontal disease, human neutrophil collagenase was purified and two monoclonal antibodies against this enzyme were obtained. This enzyme was purified by four step-affinity chromatography: heparin-aminocellurofine, gelatin Sepharose 4B, collagen-Sepharose and collagenase inhibitor column chromatographies. To produce the monoclonal antibody against the enzyme, the Balb/c mouse was immunized and its spleen cells were fused with the mouse myeloma cells. Two monoclonal antibodies to the enzyme, 2F3 (IgG1) and 3F12 (IgG1), which recognized a conformational structure of the enzyme apart from its catalytic site were obtained. Both antibodies were monospecific to leukocyte collagenase and did not cross-react with the other metalloproteinases such as leukocyte gelatinase, skin fibroblast collagenase, gelatinase and stromelysin. Using these monoclonal antibodies, collagenase was stained granularly in gingival crevicular neutrophils.

Dynamic light scattering studies on hydrodynamic properties of fibrinogen-fibronectin complex.

A high molecular weight 'cryogel' was obtained as insoluble complexes by cold incubation at near-freezing temperatures from heparinized plasma of patients with rheumatoid arthritis. After the cryogel was solubilized at 37 degrees C, 1:1 complex of fibrinogen and fibronectin was purified at room temperature by affinity chromatography on a gelatin-Sepharose 4B. Hydrodynamic properties of the complex were investigated as a function of temperature and NaCl concentration using a dynamic light scattering. The diffusion coefficients of the complex at 20 degrees C decreased with increasing of NaCl concentration as free fibronectin. The complex appears to be a more compact form at low ionic concentration, which is associated with conformational changes of fibronectin. The diffusion coefficient of the complex at 20 degrees C in 0.05 M TrisHCl(pII7.4) containing 0.5 M NaCl was estimated as 8.5 x 10(-8) cm2s-1. The complex did not dissociate over the temperature range from 20 to 37 degrees C. The diffusion coefficients of the complex decreased significantly at 12 degrees C and 40 degrees C. The thermal denaturation of fibrinogen molecule in the complex was observed at 40 degrees C. The CONTIN analysis of the light scattering data showed that the complex associated to form higher aggregates at 15 degrees C, but not at near-freezing temperature. The equilibrium between the complex and higher aggregates appeared reversible.

Clinical efficacy of topical homologous fibronectin in persistent corneal epithelial disorders.

The clinical efficacy was investigated of topical homologous fibronectin on persistent corneal epithelial defects of various etiologies. Fibronectin was purified from blood bank homologous plasma by gelatin-Sepharose 4B affinity chromatography. Twenty eight eyes of twenty five patients with persistent corneal epithelial defects and sterile corneal ulcers that failed to improve with standard therapy were treated by the instillation of homologous fibronectin eyedrops 5 times a day (500 micrograms/ml). Complete reepithelialization was achieved in all patients except two eyes due to uncontrolled glaucoma and the taking of steroids. The healing time tended to be different depending on the duration of persistent corneal epithelial defects and the severity of underlying diseases. The mean +/- standard deviation duration of epithelial defect was 68.18 +/- 77.80 days. Average healing time was 42.07 +/- 17.47 days. Ocular symptoms were relieved significantly and no side effects were observed. Over an average follow-up period of about 8 months, two cases of recurrences were noted. These results show that homologous fibronectin was also effective in patients with persistent corneal epithelial defects and corneal ulcers.

Single step purification of potent antigenic protein from sparganum by gelatin-affinity chromatography

Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum), 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.

Bronchial Epithelial Cells Produce Chemotactic Activity for Bronchial Epithelial Cells: Possible Role for Fibronectin in Airway Repair

Bronchial mucosal injury initiates a complex series of repair mechanisms, one of which is reepithelialization of a denuded lumenal surface. This suggests the hypothesis that bronchial epithelial cells, the cells initially affected by bronchial injury, might be able to initiate repair of an injured area by producing a chemotactic activity for intact bronchial epithelial cells. To evaluate this, bronchial epithelial cells were prepared from bovine lung by protease digestion and cultured in medium 199 (M199) with 10% fetal calf serum (FCS) until confluence, after which the cells were rinsed with Hanks' balanced salt solution, and serum-free fresh M199 was added. This conditioned medium was then collected and used to test the chemotactic response of bronchial epithelial cells using a blindwell chamber technique. Target cells for this assay were isolated from airways by protease digestion, grown to confluence in M199 with 10% FCS, and then harvested with trypsin. Bronchial epithelial cell-conditioned medium harvested after 3 days attracted more cells (197.0 +/- 5.7 cells/10 high power fields) than did M199 without FCS alone (4.3 +/- 0.9) (p less than 0.01). Checkerboard analysis showed that the migration was chemotactic. The chemotactic activity was nondialyzable, pepsin-labile, acid-stable, heat-labile, and lipid-inextractable. The chemotactic activity accumulated in the culture medium with time. The addition of 25 micrograms/ml of cycloheximide inhibited this accumulation. Column chromatography with Sephadex G-150 revealed a single peak of chemotactic activity in the high molecular weight range. The chemotactic activity was bound to gelatin-Sepharose 4B and was eluted with 6 M urea.(ABSTRACT TRUNCATED AT 250 WORDS)

Topical fibronectin treatment in persistent corneal epithelial defects and corneal ulcers

Topical fibronectin, autologous and homologous, was used to treat nine patients (eleven eyes) with persistent corneal epithelial defects and corneal ulcers that failed to improve with standard therapy. The fibronectin was purified from autologous and homologous plasma by gelatin-Sepharose 4B affinity chromatography and administered topically, 500 micrograms/ml five times a day, for three weeks. Complete or nearly complete reepithelialization was achieved in all patients regardless of the source of fibronectin, autologous or homologous. But healing times varied. The average healing time was 41.7 +/- 14.7 days (35.7 +/- 12.4 days for autologous, 50.8 +/- 14.4 days for homologous). Ocular symptoms were relieved significantly, and no side effects were observed. Over an average follow-up period of 5.2 months, no recurrences were noted. The results showed that homologous, as well as autologous, fibronectin was effective in patients with persistent corneal epithelial defects and corneal ulcers.

The transformation-sensitive heat shock protein (HSP47) binds specifically to fetuin

The transformation-sensitive heat shock protein of Mr=47,000 (hsp47) has been shown to bind to collagen and gelatin. We examined the binding specificity of hsp47. The binding of hsp47 to gelatin-Sepharose 4B was competitively inhibited by fetuin as effectively as by gelatin or collagen, whereas a variety of other proteins tested had no effect. Fetuin-coupled Sepharose 4B was found to bind hsp47 even at high ionic strength, but the complex was dissociated at pH ≤ 5.5.

Secretion of a novel cell-adhesive protein distinct from fibronectin by mouse L.P3 cells growing in protein- and lipid-free synthetic medium.

Several cell lines growing in protein- and lipid-free synthetic medium secreted cell-adhesive protein(s) into the medium. The conditioned medium (CM) of one of these cell lines, mouse L.P3, showed the highest cell attachment-promoting activity (CPA) among them. Cell-adhesive protein(s) in the CM of L.P3 cells (L.P3-CM) were separated into two types by sequential affinity column chromatography employing gelatin-Sepharose 4B and heparin-Sepharose 4B. One was a gelatin- and heparin-binding cell-adhesive protein (GCP), and was identified as a cellular form of mouse fibronectin. The other was a gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). The CPA of GNCP preparation was effective for the cell-attachment and spreading of both epithelial and fibroblastic cells. The CPA of GNCP preparation was not blocked by the antiserum and scarcely inhibited in the presence of the synthetic cell attachment-promoting peptide Gly-Arg-Gly-Asp-Ser-Pro, a competitive inhibitor of fibronectin. This suggests that the structure of the cell-attachment site of GNCP is different from that of fibronectin. The GNCP preparation showed little cross-reactivity with anti-mouse laminin antiserum in enzyme-linked immunosorbent assay (ELISA). These results demonstrate the possibility that GNCP in L.P3-CM is a novel cell-adhesive protein distinct from fibronectin or laminin. The secretion of the two types of cell-adhesive proteins by L.P3 cells is discussed.

Plasma fibronectin promotes modulation of arterial smooth-muscle cells from contractile to synthetic phenotype

Isolated arterial smooth-muscle cells (SMCs) cultured in medium containing whole blood serum or plasma-derived serum undergo modulation from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and cessation of the ability to contract. lnstead, an extensive rough endoplasmic reticulum and a large Golgi complex are formed and, if properly stimulated, the cells start to proliferate actively and to produce extracellular-matrix components. In vivo, a similar change in the differentiated properties of SMCs appears to be an early key event in atherogenesis. The purpose of the present investigation was to try to identify plasma components that promote the modulation of the smooth-muscle phenotype. SMCs were enzymatically isolated from rat aorta and cultured in a defined, serum-free medium. The phenotypic state of the cells was determined by transmission electron microscopy, and their growth status was followed by 3H-thymidine autoradiography and cell counting. Under these conditions, Cohn fractions I (fibrinogen) and V (albumin) were found to partially support cell attachment and transition from the contractile to the synthetic phenotype, whereas fractions II–III and IV (globulins) were inactive in this respect. Analysis on adsorptive columns of gelatin Sepharo-se 4B indicated that Cohn fraction I, but not fraction V, contained fibronectin, an adhesive protein that is present in plasma and binds to fibrinogen. When seeded on a substrate of plasma fibronectin, the cells attached with high efficiency and modulated into the synthetic phenotype at a rate similar to that observed in serumcontaining medium. In the absence of exogenous mitogens, the structural transformation of the cells was not accompanied by a proliferative response. However, when exposed to platelet-derived growth factor or serum, the cells promptly started to synthesize DNA and divide. These findings indicate that plasma fibronectin is a major determinant of the phenotypic properties of arterial SMCs, and they confirm the view that phenotypic modulation is necessary, but is itself not sufficient, for the initiation of SMC growth. The experimental system outlined here should make it possible to study the control of SMC phenotype and growth separately and under defined conditions.

16] Opsonic activity of fibronectin

Publisher Summary This chapter discusses the opsonic activity of fibronectin. Plasma fibronectin is a plasma protein that has been documented for specifically binding to a variety of other macromolecules, including collagen, actin, DNA, hyaluronic acid, fibrin, and heparin. Purified plasma fibronectin can be isolated by affinity chromatography using a gelatin–Sepharose 4B affinity column. All of the techniques that are used to assess opsonic activity of fibronectin utilize gelatinized target particles—and in most cases these particles are radioactively labeled—and macrophage uptake of the particles—as mediated by fibronectin—are assessed by radioactivity. Fresh rat liver slices are incubated with solutions containing fibronectin in the presence of a radiolabeled gelatinized lipid emulsion, under defined conditions. The liver slices are then evaluated for the uptake of radioactivity. The extent of uptake is then defined as the opsonic activity of the solutions tested. It is important to develop an assay utilizing isolated macrophages to increase the sensitivity of the opsonic assay and to investigate basic mechanisms governing macrophage–fibronectin interactions.

Fibronectin promotes epithelial migration of cultured rabbit cornea in situ.

We investigated the effect of fibronectin on epithelial migration onto the stroma in cultured rabbit cornea. Rabbit plasma fibronectin was purified by affinity chromatography using gelatin-Sepharose 4B, and its purity was confirmed by SDS polyacrylamide slab gel electrophoresis. Antibody against rabbit plasma fibronectin raised in guinea pigs formed a single precipitin line against rabbit plasma and purified rabbit plasma fibronectin by Ouchterlony double diffusion test. When rabbit cornea was cut into small blocks and cultured in TCM-199 medium alone, corneal epithelial cells began to migrate on the cut edge of the corneal stroma. The addition of purified rabbit plasma fibronectin to the culture medium significantly enhanced epithelial migration. The degree of enhancement depended on the amount of fibronectin added. When guinea pig IgG anti-rabbit plasma fibronectin was added, epithelial migration was significantly inhibited when compared with that in control cultured corneal blocks. The results demonstrate that fibronectin promotes epithelial migration in the cornea and thus plays an important role in corneal wound healing.