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W Hornebeck

Publications and source records attributed to W Hornebeck.

At least 19 recordsLinked to original sources
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↗
PubMed1991

A lipophilic heparin derivative protects elastin against degradation by leucocyte elastase.

An oleolylated derivative (I) of partially N-desulphated heparin was prepared containing an average number of three oleoyl residues for one molecule of heparin. The inhibitory capacity of I (IC50 = 0.55 microM) for leucocyte elastase resembles that of heparin (IC50 = 0.2 microM). In contrast to heparin, I is also an inhibitor of porcine pancreatic elastase (IC50 = 0.68 microM) and it also has the capacity to protect elastin fibres against the degradation by leucocyte elastase. When insoluble elastin is pretreated with I its degradation by leucocyte elastase is inhibited by almost 90% while pretreatment of elastin with heparin exhibited only a moderate effect on elastolysis (10% inhibition).

Amino Acid Sequence↗
PubMed1991

Growth stimulation of human keratinocytes by tissue inhibitor of metalloproteinases.

Human recombinant tissue inhibitor of metalloproteinases (rTIMP) at 0.2-4.6 microM was found to stimulate the growth of normal human keratinocytes, in primary cultures on a plastic support, and to markedly increase their growth on a tridimensional culture system, the skin equivalent, as shown by histology, DNA measurements, and planimetry. In contrast, rTIMP had no effect on the growth of normal human fibroblasts. The growth of keratinocytes on extracellular matrix components produced by keratinocytes cultured in the presence or absence of rTIMP was similar, suggesting that rTIMP does not stimulate keratinocyte growth by modifying either the quantity or the composition of the extracellular matrix deposited. rTIMP was labeled with 125iodine in order to study its interaction with keratinocytes in culture. Binding of (125I) rTIMP to keratinocytes was found to be temperature and time dependent. Under steady-state conditions at 22 degrees C, one class of specific rTIMP binding sites was identified with KD of 8.7 nM and 135,000 sites/cell. Such findings are in keeping with the known potentiating effect of TIMP on erythroid precursors, and indicate that this protein has at least two distinct activities.

Binding Sites↗
PubMed1991

The elastolytic activity of cathepsin G: an ex vivo study with dermal elastin.

To determine whether human neutrophil cathepsin G can act by itself or in concert with human neutrophil elastase to destroy elastic fibers in vivo, we used cryostat sections of human skin as an ex vivo substrate for these leukoproteinases. Specifically stained dermal elastic fibers were quantitated using an accurate and almost entirely automatic morphometric procedure that included computerized threshold selection and elimination of non-elastic dark elements. AA, the area fraction occupied by the dermal elastic fibers, was found to be 0.100 +/- 0.014 (mean +/- SD) for 21 control skin sections originating from a single donor. Measurement of the fiber diameters in these control sections (2.4 +/- 0.8 microns [mean +/- SD]) allowed calculation of the Weibel factor used to convert AA into Vv, the volume fraction occupied by the elastic fibers: Vv was 0.028 +/- 0.004 (mean +/- SD). Incubation of skin sections with elastase, cathepsin G, or mixtures of the two enzymes resulted in an important decrease in AA accompanied by a slight increase in the average fiber diameter. The largest increase (14%) was noticed for cathepsin G and was due to a preferential attack of thin fibers and to fiber fragmentation. The AA of fibers remaining after elastolytic activity of cathepsin G was 20 to 30% that of elastase in this ex vivo assay. On the other hand, cathepsin G stimulated the elastolytic activity of elastase. For instance, the activity of a mixture of 1.1 microM elastase and 1.5 microM cathepsin G was 1.9-fold higher than the sum of the activities of the individual proteinases. The stimulation increased with the cathepsin G concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Characterization of metalloelastase-like activity from the plasma of a patient with Tangier disease.

An enzymatic activity with releases p-nitroaniline from 3-carboxypropionyl-trialanine p-nitroanilide (Suc[Ala]3NA) was characterized in blood plasma of patients with Tangier disease. This activity results from the sequential action of a metalloendopeptidase (MP) and an aminopeptidase (AP). These proteases were purified 134- (MP) and 82-fold (AP) from low density and very low density lipoproteins (LDL and VLDL) depleted Tangier plasma by DEAE-Trisacryl chromatography and gel filtration. MP and AP could be separated by polyacrylamide gel electrophoresis. MP shares some analogy with neutral endopeptidase (membrane metalloendopeptidase, EC 3.4.24.11) and is able to degrade human plasma fibronectin (mainly to fragments of 185, 168 and 128 kDa) as evidenced on Western blots. It cannot hydrolyse 3H-labelled insoluble elastin and apolipoprotein AII, but did cleave a dinitrophenyl-octapeptide as well as apolipoprotein AI to 25-kDa and 24-kDa fragments formed sequentially. It may therefore be partially responsible for the in vivo degradation of apoAI observed in Tangier disease.

Amino Acid Sequence↗
PubMed1991

Prevention of leucocyte elastase-induced emphysema in mice by heparin fragments.

Heparin and its derivatives inhibit human leucocyte proteinases i.e. elastase and cathepsin G, but do not inhibit porcine pancreatic elastase and Pseudomonas aeruginosa elastase. In vitro experiments, reported here, also indicate that elastin, one of the physiological substrates of human leucocyte elastase (HLE), could decrease by 30-fold the inhibitory potential of an hexadecasaccharide heparin fragment (dp 16) isolated from CY 222. Nevertheless, the inhibitory capacity of the heparin fragment still remains elevated with IC50 = 2.7 x 10(-7) M and still inhibits HLE in its free and adsorbed state to elastin. These overall data prompted us to evaluate the influence of CY 222 in HLE-induced emphysema. Emphysema was induced in mice eight weeks old, following a single instillation of 200 micrograms of HLE. CY 222 treated animals received 2.5 mg.kg-1 subcutaneously once daily, 6 days per week during 4 weeks prior to HLE instillation, and for eight weeks following HLE instillation. The heparin fragment treatment of the mice halved the mortality rate observed early following HLE instillation. After 8 weeks, surviving animals were examined for lung histological and morphometrical changes: mean linear intercept (MLI) and internal alveolar area (ISA). The CY 222 heparin fragments exerted a protective effect against HLE-induced emphysema by decreasing by 70% the MLI; these heparin fragments exerted no effect on emphysema induced by pancreatic elastase in hamsters or mice. Heparin derivatives represent a new class of physiological HLE low molecular weight inhibitors capable of preventing HLE-induced emphysema.

Animals↗
PubMed1991

[Matrix metalloproteinase (MMP)].

Matrix metalloproteinases (MMP) are a group of zinc endopeptidases responsible for the degradation of most extracellular matrix macromolecules as observed during many physiological and pathological processes; their role in tissue remodeling with aging is postulated. Members of this family share high level of structural analogy and are secreted by several cell types as zymogens. Many factors (cytokines, chemicals, growth factors, cell aging...) are able to interfere with MMP's expression at the transcriptional level. The activation of MMP's zymogens can be catalyzed by different agents: proteinases, Hg salts, disulfides, superoxide ions... Within the extracellular space, the activities of MMPs are controlled by a family of inhibitors: the tissue inhibitors of metalloproteinases or TIMPs.

Enzyme Precursors↗
PubMed1990

Susceptibility of baboon aorta elastin to proteolysis.

Elastin was purified from baboon aorta using Achromobacter collagenase and its susceptibility to proteolysis by various enzymes was studied. Human leukocyte elastase (HLE) hydrolysed baboon aortic elastin 8 times faster than human cathepsin G. Bovine chymotrypsin had virtually no activity against this substrate. The kinetic constants V and [S50] of aortic elastin hydrolysis by HLE (0.15 microM) were 0.00286 mg x ml-1 x min-1 and 0.158 mg x ml-1, respectively. One mg of this elastin could be saturated with 5.6 micrograms of HLE. As with elastins isolated from other sources, the hydrolysis of baboon aortic elastin by HLE was highly sensitive to ionic strength, and a biphasic effect was obtained with increasing NaCl concentrations. A nearly 2-fold stimulation of elastolysis was observed at a 0.15M NaCl concentration. Further increase in ionic strength led to a continuous decrease of the rate of elastolysis which paralleled the decrease of adsorption of elastase to baboon aortic elastin. Cathepsin G, but not bovine alpha-chymotrypsin, was able to stimulate the rate of hydrolysis of baboon aortic elastin by HLE. A 1.7 fold stimulation was observed for a 1:1 molar ratio of the two proteinases and rose to 2.1 for a HLE/Cat. G ratio equal to 8.

Animals↗
PubMed1990

[Lipoproteins, elastases and atherogenesis].

Increased lipid deposition and fragmentation of elastic lamellae are main events in atherogenesis. Relationships between lipoproteins and elastase can exist: i) interactions between lipoproteins and arterial wall cells from mesenchymal or inflammatory origin can modulate the expression and secretion of elastase; ii) such neutral proteases can degrade protein constituents of lipoproteins, and modify their physical and biological properties.

Animals↗
PubMed1990

[Endothelial cell proteases and their modulation by platelets].

Endothelial cells produce and secrete a large number of proteases which are implicated in various disease states. These proteases fall into two classes: serine proteases include plasminogen activators (t-PA) and urokinase (u-PA) and play a major role in fibrinolysis, tissue repair and carcinogenesis; and metalloproteases include collagenases and stromelysine, two enzymes involved in the tissue remodelling that occurs during angiogenesis and tumor growth. The authors have recently identified two other proteases in porcine aortic endothelial cell culture medium. One is an elastase-like enzyme of the metalloprotease group, whereas the other is a new protease whose molecular weight is 85 Kd and whose activity becomes apparent only after exposure of the endothelial cells to platelets. The term Platelet Endothelial Cell Activated Protease accurately describes this enzyme. PECAP degrades casein and fibrinogen. Because PECAP is not inhibited by the usual inhibitors of the various classes of proteases, it remains at present unclassified.

Blood Platelets↗
PubMed1990

[Stimulation of epidermal growth in equivalent skin by tissue inhibitor of metalloproteases].

Tissue inhibitor of metalloproteases (TIMP) is a major regulator of extracellular matrix synthesis and degradation. Moreover elevated tissue inhibitor of metalloproteases levels are found in the blister fluid of many diseases, suggesting that tissue inhibitor of metalloproteases plays a role in epidermal and dermal wound healing. The aim of this work was to study the effects of human recombinant tissue inhibitor of metalloproteases in a skin equivalent model used to evaluate the effects of drugs on epidermal and dermal components. Planimetry, DNA assays, and histologic studies showed that tissue inhibitor of metalloproteases enhanced the growth of normal human keratinocytes on dermal equivalent. Thus, the extracellular matrix regulator tissue inhibitor of metalloproteases also stimulates the growth of keratinocytes. Identity of the gene encoding tissue inhibitor of metalloproteases and Erythroid Potentiating Activity (EPA) has been reported. The effects of tissue inhibitor of metalloproteases on keratinocytes described herein are reminiscent of the stimulating effect of erythroid potentiating activity on the growth of erythroid precursors.

DNA↗
PubMed1989

Elastase-like activity in cultured aortic endothelial cells.

Cultured porcine aortic endothelial cells were studied for cellular and secreted elastase activity. We describe an activity hydrolyzing the synthetic elastase substrate, succinyl(alanine)3 nitroanilide, but not elastin, which was shown to be membrane located and was not secreted to the culture medium. A different neutral proteinase activity degrading insoluble elastin was demonstrated in the culture medium following its fractionation by gel filtration high performance liquid chromatography (HPLC). Since no elastinolytic activity could be directly detected in the conditioned medium, it is likely that the chromatographic separation removed an endogenous inhibitor.

Animals↗
PubMed1989

Interaction of platelets with endothelial cells: activation of a novel neutral protease.

The activation of a new neutral protease of MW 85,000 was demonstrated on interaction between porcine aortic endothelial cells and human and porcine platelets in culture. The activity of this enzyme, PECAP (platelet endothelial cell activated protease), was detected by electrophoresis on polyacrylamide gels impregnated with the substrate casein. Our results showed that the platelet-endothelial cell interaction did not involve induction of synthesis of de novo enzyme, but rather an activation of a latent enzyme. PECAP cleaved casein and fibrinogen, but had no activity against gelatin or elastin. It was not inhibited by inhibitors of metalloproteases (EDTA, 1.10 phenanthroline), serine proteases (phenylmethylsulfonyl fluoride, elastatinal), or cysteine proteases (iodoacetate, N-ethylmaleimide) and it seems to be unrelated to the previously known proteases of mesenchymal and hematopoietic cells.

Animals↗