Search PubMed⌕ Search

Biomedical subjects

W Hornebeck

Publications and source records attributed to W Hornebeck.

At least 73 records · Page 4Linked to original sources
PubMed1994

Fibrinolysis potentiating activity following endothelial cells-platelet interaction.

When porcine endothelial cells in culture are incubated in the presence of human platelets, a 90kDa neutral proteinase activity is generated on casein gel (PECAP-Platelet Endothelial Cell Activated Protease). This activity was undetected when platelet extract or serum free EC conditioned medium were analysed under similar conditions. The optimum pH, isoelectric point, molecular weight and inhibitory profile of this activity were similar to Glu-plasmin. However, the low plasminogen content (less than 50ng/ml) in the conditioned medium of endothelial cells incubated with platelet could not contribute alone to this activity and the presence of a plasmin potentiating factor was suggested. This factor was separated from plasminogen by lysine-Sepharose chromatography.

Animals↗
PubMed1994

Tenascin distribution in articular cartilage from normal subjects and from patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To determine whether tenascin is present in normal and diseased human cartilage. METHODS: Immunohistochemical and biochemical assays with a monoclonal antibody against all tenascin isoforms (BC-4) were used. RESULTS: Cartilage samples from osteoarthritis and rheumatoid arthritis patients contained increased amounts of tenascin compared with the levels in normal cartilage. Human fetal cartilage was also found to contain tenascin. In normal cartilage explants treated with interleukin-1 beta, tenascin was present in pericellular areas of all layers. Immunolocalization studies revealed that tenascin was most abundant in the superficial layers of osteoarthritic cartilage. Western blot analysis performed from dissociative extracts of diseased cartilage confirmed the presence of subunits of the native molecule. CONCLUSION: Tenascin is increased in arthritic cartilage and is weakly expressed in normal cartilage.

Adult↗
PubMed1994

Modulatory effect of dexamethasone on ornithine decarboxylase activity and gene expression: a possible post-transcriptional regulation by a neutral metalloprotease.

The intracellular effect of dexamethasone (DXME) on the activity and gene expression of ornithine decarboxylase (ODC) was studied in Syrian hamster embryo cells (SHE). The ODC activity (expressed as nmoles decarboxylated ornithine mg-1 protein h-1) was 4.61 +/- 0.14 in untreated cells, whereas it increased to 14.38 +/- 0.26 after 5 h treatment with 1.6 x 10(-7) M TPA. In contrast, DXME (2.5 x 10(-5) M) reduced the ODC activity by 50 per cent to 2.35 +/- 0.22. In cells co-treated for 5 h with TPA and DXME, ODC activity decreased to the level of the untreated cells. However, when DXME was added 3 h after TPA treatment for 2 h, in the continuous presence of TPA, the ODC activity unexpectedly increased further to 16.44 +/- 1.05. The modulation of ODC activity correlated partly with the level of ODC mRNA. Thus when cells were treated with TPA, the ODC mRNA increased threefold, whereas it decreased by 30 per cent when the cells were exposed to DXME. In TPA-DXME co-treated cells, as in TPA pretreated cells followed by DXME for 2 h, a decrease (31.25 per cent and 12.5 per cent respectively) was observed in ODC mRNA. In turnover studies, DXME was found to increase the stability of ODC; the discrepancy between ODC activity and ODC mRNA levels could result from an inhibitory effect of the corticoid on proteolysis of ODC. Studies of lysosomal protease showed that the activities of cathepsins L, B and H decreased following TPA treatment. DXME also inhibited cathepsin L and B activities, but stimulated cathepsin H.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effect of hyaluronan on the elastase-type activity of human skin fibroblasts.

The influence of hyaluronan (HA) on the expression of human skin fibroblast elastase-type protease (HSFEp) (Homsy et al, 1988) was studied. At confluency of HSF cultures, hyaluronan increased the level of HSFEp in a time and dose-dependent fashion. Optimal effect was observed after 48 h of culture and at 2 mg/ml HA concentration; the stimulatory effect of HA could be suppressed by 1 microM cycloheximide. The enhancement of enzyme biosynthesis by HA was dependent on cell proliferation but quasi invariant with HSF passage number (from 7-21).

Cell Division↗
PubMed1994

Increased secretion of latent elastase activity following contact between human skin fibroblasts and elastin derived peptides.

Elastin-derived peptides were previously shown to influence human skin fibroblasts (HSF) chemotaxis and proliferation (Ghuysen et al., 1992). We report here that culturing HSF on kappa-elastin (kappa E) but not onto fibronectin (FN) enhanced the secretion of latent elastinolytic activity. The proteinase involved was identified as the 72 kDa gelatinase A. Moreover, HSF-kappa E as well as HSF-FN interactions modulated the secretions of Il1 induced expressions of elastinolytic activities.

Adolescent↗
PubMed1994

Presence of gelatinase A and metalloelastase type protease at the plasma membrane of human skin fibroblasts. Influence of cytokines and growth factors on cell-associated metalloendopeptidase levels.

Gelatinase A and elastase type proteinase (Homsy, et al., 1988) present at plasma membranes of human skin fibroblasts (HSF) were separated by anion exchange chromatography on a DEAE Tris acryl M column. Elastase type proteinase (HSFE1) was able to convert 72 kDa progelatinase A to a lower 66 kDa M.W. active enzyme. Several cytokines (IL-1 beta, IL4, IL6), interferon gamma (IFN gamma) and tumor growth factor beta (TGF-beta) were studied for their ability to modify the levels of those plasma membrane associated proteinases. Among these mediators, only IL-1 beta was found to enhance the amounts of HSF membrane-bound HSFE1 and Gelatinase A.

Cell Membrane↗
PubMed1994

An animal model for the assessment of gingival lesions.

A model of gingival inflammation was performed in Sprague-Dawley rats weighing 180-200 g. Mechanical bamboo stick-induced injury was inflammatory when bacteria contaminated the sticks. Bacteria were first obtained from gingival fluid collected from a patient with adult periodontitis. Another strain from Institut Pasteur (IP 6444) induced similar inflammation. Inflammation was then quantified 10 days later by means of elastase assays performed in gingival extracts. In parallel, elastic structures were observed and elastic fibers were quantified by automated image analysis. This technique of "impaction" was able to induce a gingival inflammatory reaction characterized by a significant increase of gingival elastase content, infiltration of gingival tissues by elicited cells, and gingival elastic fiber breakdown. These parameters were correlated, and measurement of one of them might be useful for pharmacological studies applied to the treatment of periodontal lesions. An example of results obtained from animals treated by heparine was shown.

Animals↗
PubMed1994

Heparin and its derivatives modulate serine proteinases (SERPS) serine proteinase inhibitors (SERPINS) balance. Physiopathological relevance.

Heparin and heparan sulfate, exhibiting wide biological interactions, are constituted of block structures. A defined pentasaccharide motif was found responsible for the enhancement of the rate of inactivation of factor Xa by antithrombin III. Heparin also interacts with other serine proteinase inhibitors as protease nexin I, and thus possibly modulates extracellular matrix proteolysis by serine proteinases in the pericellular environment. Human neutrophil elastase (HNE) activity is inhibited by heparin with Ki = 75 pM. This strong interaction is electrostatic, involving HNE/arginine residues disposed in a "cluster shoe" arrangement on the surface of the molecule and mainly OSO3- groups of heparin. HNE-heparin interactions also interfere with HNE associations with its natural inhibitors: it decreases the rate of association of HNE with alpha 1 proteinase inhibitor (alpha 1 P(i)) by 3 orders of magnitude, while increasing kass between HNE and mucus bronchial inhibitor (MBI) by > 10 fold. In vivo experiments demonstrated that heparin fragments lacking anticoagulant activity were able to nearly completely abolish emphysematous lesions induced in mice by a single intratracheal administration of 200 micrograms HNE. Long chain unsaturated fatty acids peptide conjugates were described as competitive HNE inhibitors (Hornebeck W. et al. 1985). We synthesized N-oleoyl heparin derivative (3 oleoyl groups/one molecule of heparin); such a lipophilic glycosaminoglycan (LipoGAG), although acting as an elastin protecting agent, possessed lower HNE inhibitory capacity as compared with heparin. In contrast, however, it was able to inhibit other serine proteinases such as urokinase, plasmin, porcine pancreatic apha-chymotrypsin and elastase. Such Lipo GAG's can be therefore useful to control matrix metalloproteinases (MMPs) during tissue remodeling or tumor invasion.

Animals↗
PubMed1994

Retarded fibronexus formation and cell attachment on type V collagen.

We previously found that type V collagen repressed the attachment and spread of aortic smooth muscle cells. The present study was carried out to investigate the effects of type V collagen on the formation of fibronectin and F-actin filaments of human dermal fibroblasts in relation to cell attachment and spread, using an immunofluorescent technique and morphometry. The number and area of the cells attached to type V collagen at 1 and 3 hours after seeding were significantly lower than those of cells on other substrates, including collagen types I, III and IV, and bovine serum albumin. However, there was no significant difference in the attachment and spread among the cells on these substrates after 24 hours. Cultured fibroblasts exhibited two patterns of fibronectin; one was a clear, linear fibronectin localized mainly in the cellular margins, and the other was a granular or flocculent fibronectin found in the perinuclear areas. The former was stained in non-permeabilized cells, but not in trypsin-treated cells (cell surface fibronectin). In contrast, the latter was not detected in nonpermeabilized cells, but was found in trypsin-treated cells (perinuclear fibronectin). Most of the cells cultured on type V collagen did not form either linear cell surface fibronectin or F-actin filaments at 3 hours. In contrast, many cells on collagen types I, III, and IV developed both cell surface fibronectin and F-actin filaments, the distributions of which were partially coincident. Colocalization of linear cell surface fibronectin and F-actin filaments was found in cells on all of the substrates after 24 hours. Perinuclear fibronectin showed similar patterns, and was not colocalized with F-actin filaments on different substrates at 3 and 24 hours of culture. Solid-phase substrates induced a better cellular attachment at 3 hours than serum adhesive factors. The administration of monensin, which inhibits the secretion of protein products, decreased the intensity of the fluorescence of cell surface fibronectin in fibroblasts, which was observed in a clear line. These results suggest that the retardation of the initial attachment and spread of fibroblasts on type V collagen is related to an inhibition in the formation of the fibronexus, a close transmembranous association of individual fibronectin fibers and F-actin filaments.

Actins↗
PubMed1993

Inhibition of the human leukocyte endopeptidases elastase and cathepsin G and of porcine pancreatic elastase by N-oleoyl derivatives of heparin.

N-oleoyl-heparin derivatives differing in their oleic acid and sulfate contents were synthesized and studied for their abilities to inhibit human leukocyte elastase (HLE), human leukocyte cathepsin G (CatG) and porcine pancreatic elastase (PPE) at pH 8.0, ionic strength 0.05 M and 37 degrees. Heparin (Hep) as well as N-oleoyl-heparins behaved as tight-binding, hyperbolic noncompetitive inhibitors of HLE (KiHep = 75 pM) and CatG (KiHep < 25 pM). The main driving force for the interaction between enzymes and glycosaminoglycans was electrostatic in nature. Under the condition [enzyme] >> Ki, the stoichiometries of the interaction with Hep were 1:2 (Hep:HLE) and 1:4 (Hep:CatG). Coupling one oleic acid residue to three disaccharide units of partially N-desulfated Hep, Ol1:3Hep, lowered HLE inhibition (Ki = 0.3 nM) and the stoichiometry of binding was reduced to 1:1. Re-N-sulfation of a similar derivative, Ol1:5Hep(SO4), containing one fatty acid residue for five disaccharide units, led to a substance with similar HLE inhibitory characteristics as Hep (Ki = 92 pM) and stoichiometry 1:2. Ol1:5Hep(SO4) was also a more efficient inhibitor of CatG (Ki < 33 pM) than Ol1:3Hep (Ki = 9.5 nM). The residual activities of N-oleoyl-Hep complexes with CatG were much lower than the corresponding activities in the presence of Hep. While oleate and Hep could not inhibit PPE, N-oleoyl-Hep, independently of fatty acid substitution and sulfate content, could inhibit this enzyme with Ki congruent to 60 nM and low residual activity. The efficient endopeptidase inhibitory characteristics of N-oleoyl-Hep derivatives, together with their non-anticoagulant properties and their capacity to interact with elastin, may be therapeutically useful in connective tissue degenerative diseases.

Amino Acid Sequence↗
PubMed1993

Inhibition of human leucocyte elastase by fatty acyl-benzisothiazolinone, 1,1-dioxide conjugates (fatty acyl-saccharins).

Derivatives of benzisothiazolinone 1,1-dioxide (saccharin) N-acetylated with aliphatic and aromatic substituted aliphatic acyl groups were prepared. The inhibitory activity of the compounds was assayed against human leucocyte elastase (EC 3.4.21.37) and several other proteases. The IC50 values for inhibition of the human leucocyte elastase decreased with increasing length of the acyl residue, and reached a minimum value at C16 (2 microM). This phenomenon and the decrease of the inhibition by surfactants or by saturation of the enzyme with palmitic acid, indicates that in addition to acylation, hydrophobic interactions are also involved in the inhibition of this proteinase by compounds substituted with acyl groups containing at least 12 carbon atoms. The inhibitory activity of N-palmitoyl-benzisothiazolinone 1,1-dioxide (palmitoyl-saccharin) is about 14 times higher toward human leucocyte elastase than for thrombin (EC 3.4.21.5), and several hundred times, compared to porcine pancreatic elastase (EC 3.4.21.36) and to plasmin (EC 3.4.21.7). Fatty acylated saccharin derivatives were seen to bind in a saturable fashion to insoluble elastin, and decreased the susceptibility of this protein to hydrolysis by human leucocyte elastase.

Amino Acid Sequence↗
PubMed1993

Protective effect of oleoyl peptide conjugates against elastolysis by neutrophil elastase and kappa elastin-induced monocyte chemotaxis.

Elastin can impair the human neutrophil elastase (HNE) inhibitory capacity of elastase inhibitors. We synthesized oleoyl-alanyl-alanyl-prolyl-valine (Ol-Ala-Ala-Pro-Val-OH) (oleoyl peptide) and the amides (NH2 and NH-C3H7) of this peptide and studied their HNE-inhibitory potencies using succinyl-alanyl-alanyl-alanine-p-nitroanilide (Suc-Ala-Ala-Ala-pNA) or 3H-labeled elastin as substrates, as well as cryostat sections of rabbit skin as an ex vivo substrate. Using Suc-Ala-Ala-Ala-pNA, Ol-Ala-Ala-Pro-Val-OH had an IC50 of 3 microM. When the COOH terminal of the oleoyl peptide was derivatized to amide forms, the compound lost its ability to interact with HNE while keeping its elastin-protecting function: IC50 values for NH2 and NH-C3H7 derivatives were 22 and 17 microM, respectively. Also, the HNE-inhibitory capacity of Ol-Ala-Ala-Pro-Val-OH was only reduced 2-fold by using elastin as a substrate. This decrease was much lower than those determined with other HNE inhibitors of similar potency and could be accounted for by the ability of oleoyl peptide to bind to elastin. Cryostat sections of rabbit skin were also used as an ex vivo substrate for assessing the elastin-protecting property of Ol-Ala-Ala-Pro-Val-OH. Preincubating HNE and oleoyl peptide before application to tissue sections led to an IC50 of 8 microM, close to the value determined with elastin as a substrate. Treatment of sections with oleoyl peptide before adding HNE gave a lower IC50 (4 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

[Tissue inhibitors of matrix metalloproteinases TIMP 1-2. Structures and functions].

Tissue inhibitors of matrix metalloproteinases (TIMPs) represent a family of ubiquitous proteins which main biological function resides in their ability to control the activity of matrix metalloproteinases (MMPS). The structures and specificities of TIMP1 and TIMP2 are described. Moreover, TIMP1, but not TIMP2, is able to stimulate the growth of several cell types. TIMP1 interacts with keratinocytes via a receptor mediated pathway (Kd = 8.7 nM; 135,000 sites/cell). Thus, this inhibitor does contain several structural domains able to recognize i) MMP active site, ii) pro-MMP9 hemopexin-like domain, iii) cell membrane proteins.

Animals↗
PubMed1992

Predictions of the secondary structure and antigenicity of human and bovine tropoelastins.

Secondary structure and antigenicity predictive methods have been applied to the sequences of human and bovine tropoelastins in order to have some insight into the molecular structure of its insoluble counterpart, i.e., elastin. For both tropoelastins, all the predictions yielded 11 major regions, in which the pleated conformation was predominant, separated by 10 strong helical segments of various lengths located within alanyl rich regions of the chains. The overall conformations of human and bovine tropoelastins were estimated to contain 18 +/- 5% alpha-helices, 63 +/- 17% beta-sheets, 13 +/- 13% beta-turns and 6 +/- 6% random coil. For both tropoelastins, antigenicity predictions indicated the presence of seven synthetic decapeptides corresponding to continuous linear epitopes of the molecule. Some of the predicted epitopes are located in the same regions in both species while others are not. These predictions have allowed us to propose an alpha/beta conformation for tropoelastin. Therefore this extracellular matrix macromolecule might be more structured (10 helical segments for about 18% of the overall structure) than previously suggested.

Algorithms↗
PubMed1992

[Inhibition of elastase activity in human gingival extracts by elastin peptides].

Effects of elastin peptides on elastase activity of gingival biopsy specimen extracts were studied in vitro and in vivo. All the extracts from biopsy specimens from 15 patients with a variety of periodontal diseases demonstrated fairly marked elastase activity. In vitro, addition of elastin peptides produced a mean inhibition of 54% +/- 14% with a concentration of 0.25 mg/ml and a mean inhibition of 90% +/- 6% with 2.5 mg/ml. In vivo treatment of gums with a paste containing 1% elastin peptides reduced elastase activity by 47% in 7 of 8 patients. These data suggest that elastin peptides are effective inhibitors of periodontal elastase activity and may be useful in preparations used for the preventive or curative treatment of periodontal diseases with tissue lysis.

Adolescent↗
PubMed1991

Mechanisms of interaction between human skin fibroblasts and elastin: differences between elastin fibres and derived peptides.

3H-Labelled kappa-elastin peptides (kE:75 kDa molecular weight) were shown to bind to confluent human skin fibroblast (HSF) cultures in a time-dependent and saturable manner. Scatchard analysis indicated the presence of high affinity binding sites with kD = 2.7 x 10(-10) M and 19,000 sites per cell. Binding of kE to its receptor on HSF accelerates and intensifies the adhesion of insoluble elastin fibres (iE) to confluent HSF. Optimal effect was attained for a kE concentration of 0.3 x 10(-9) M close to kD. This stimulatory effect of kE on the binding of iE to HSF could be inhibited by neomycin, retinal and pertussis toxin, substances which act at different levels of the transduction mechanism following the activation of the receptor and the subsequent triggering of cell biological events (chemotaxis, modification of calcium fluxes). The stimulation of iE adhesion to HSF induced by kE as well as kE binding to the cells could be inhibited by lactose and laminin but not by Arg-Gly-Asp-Ser(RGDS) peptides. This indicates that the elastin peptide receptor on HSF possesses lectin-like properties and shares homology with the laminin receptor as also shown for other cell types. None of the substances tested, that is inhibitors of the transduction mechanism, lactose, laminin and Arg-Gly-Asp-Ser(RGDS) peptides were shown to interfere significantly with the binding of iE (in the absence of added kE) to confluent HSF. The proteins adhering strongly to elastin fibres were isolated by a sequential extraction procedure and the final hydrochloride guanidinium-DTT extract was analysed by SDS-PAGE under reducing conditions, Western blots using specific antibodies against several connective tissue proteins and affinity for [3H]-kE following nitrocellulose electro-transfer of proteins. Fibronectin, vitronectin, tropoelastin(s), and a 120 kDa cysteine rich glycoprotein previously designated as elastonectin were identified. Among these proteins, [3H]-kE was found to bind exclusively to a 65 kDa protein that could be eluted selectively from elastin fibres with a neutral buffer containing 100 mM lactose. Therefore the elastin peptide receptor on human skin fibroblasts shares properties with the elastin receptor characterized from other cell types. Conformational differences between elastin peptides and elastin fibres could explain the differences in the mechanisms of interactions between elastin fibres and elastin peptides with HSF in culture. The stimulatory effect of elastin-derived peptides on the adhesion of elastin fibres to HSF could have implications in the oriented biosynthesis of elastin fibres.

Adult↗
PubMed1991

Interaction between elastin and tumor cell lines with different metastatic potential; in vitro and in vivo studies.

Interactions between the extracellular matrix macromolecules and tumor cells are critical in the process of metastasis formation. We show here that elastins (both mature insoluble elastin and a 75-kDa soluble peptide: K-elastin) adhere rapidly to two cell lines with high metastatic capacities: a metastatic lung carcinoma cell line (3LL-HM) and a human amelanotic melanoma cell line (A-2058); by contrast the low-metastatic Lewis lung carcinoma cell line variant as well as a rhabdomyosarcoma cell line with a low metastatic potential bind to elastins to a much lower extent. 3H-labelled K-elastin was used in order to study elastin--3LL-HM interaction. It was found to be saturable (2 ng 3H-labelled K-elastin/10(6) cells), with one class of high-affinity binding sites having Kd equal to 1.3 nM and 16,000 sites/cell. The binding of K-elastin to 3LL-HM cells at its receptor triggered several cell responses; (a) increase of intracellular Ca2+ concentration; (b) induction of 3LL-HM chemotaxis toward the K-elastin gradient; (c) stimulation of the adherence of mature insoluble elastin. In contrast to non-transformed cells such as fibroblasts and smooth muscle cells, the adhesion kinetics of insoluble elastin to 3LL-HM did not exhibit a lag period; the rapid binding of insoluble elastin to the tumor cells was followed by its slow detachment from the cells, which lasted for 6 h. 3LL-HM cells but not human skin fibroblasts were shown to secrete elastinolytic activity inhibitable by metal-chelating agents. In vivo studies were performed in order to evaluate the influence of K-elastin binding to 3LL-HM cells on their ability to form lung colonies in mice. It was shown that pretreatment of 10(4) 3LL-HM cells with 10 microM K-elastin and the simultaneous i.v. injection into mice of 750 micrograms K-elastin together with the highly metastatic cells was able to reduce the number of lung colonies by more than 70% after 12 days.

Adult↗