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Biomedical subjects

L Robert

Publications and source records attributed to L Robert.

At least 181 records · Page 10Linked to original sources
PubMed1987

Prevention by calcitonin of the pathological modifications of the rabbit arterial wall induced by immunization with elastin peptides: effect on vascular smooth muscle permeability to ions.

Immunization of rabbits with elastin peptides prepared from purified bovine ligamentum nuchae elastin produces calcified arteriosclerotic lesions and fragmentation of elastic lamellae. Simultaneous administration of porcine calcitonin largely prevents the development of lesions. Experiments were carried out to clarify the mechanisms involved in the development of lesions as well as those involved in the preventive effect of calcitonin. Control experiments were carried out using bovine serum albumin (BSA) as antigen. Circulating antibodies and soluble immune complexes increased steadily in the sera of animals immunized with elastin peptides or BSA. The cellular immune reaction was weak as assessed by [3H]thymidine incorporation into lymphocytes in the presence of antigen or phytohemagglutinin. Arterial lesions appeared only in the animals immunized with elastin peptides, not in those immunized with BSA. Ion flux measurements were also carried out on strips of aorta obtained from immunized and control animals. Immunization with elastin peptides significantly increased the ouabain-insensitive 22Na+ efflux, the 86Rb efflux (indicator of K+ efflux), and the 45Ca2+ influx. Simultaneous calcitonin administration prevented the increase in Ca2+ influx but did enhance passive permeability to Na+ and K+ as well as the sodium pump. When calcitonin was administered without immunization, it decreased arterial smooth muscle permeability to Na+ and K+ and also decreased the basal Ca2+ influx. It is concluded that the pathological modifications of the arterial wall triggered by immunization with elastin peptides is at least partly mediated by the effect of antielastin antibodies and immune complexes on the ion permeability of arterial smooth muscle. Prevention of the increased Ca2+ influx by calcitonin is probably a key effect in the prevention of the development of lesions. The fact that calcitonin alone can modify the ion permeability of arterial smooth muscle suggests that this hormone may play a role in the regulation of vascular homeostasis.

Animals↗
PubMed1987

Immunology of elastin: study of anti-elastin peptide antibodies by DOT immunobinding assay.

In order to further investigate the role of the immune system in the arteriosclerotic process, we investigated the anti-elastin peptide antibodies (AEAb) of the IgG and IgM types by DOT immunobinding assay in the sera of patients suffering from various arteriosclerotic diseases. In total 232 control and pathological sera were studied. In obliterative arteriosclerosis of the legs 90%, ischemic heart disease 67% and hypertension 60% of sera were positive for AEAb of the IgG type independent of age. In the case of diabetes mellitus, however, the duration of the disease was determinant. In rheumatoid arthritis, the results were negative. No clear-cut positivity could be demonstrated in stroke patients either. These results indicate that AEAb can be detected in some diseases and DOT appears to be an appropriate method for the AEAb screening in various diseases.

Adult↗
PubMed1987

Effect of elastin peptides on ion fluxes in mononuclear cells, fibroblasts, and smooth muscle cells.

Elastin peptides prepared by alcoholic potassium hydroxide degradation of highly purified fibrous elastin from bovine ligamentum nuchae (kappa-elastin) were shown to act on the ion channels of human monocytes, aorta smooth muscle cells, and skin fibroblasts. In small amounts (between 0.1 and 1 microgram/ml), elastin peptides strongly increased calcium influx and inhibited calcium efflux by an apparently calmodulin-dependent mechanism. They also were shown to increase sodium influx and to decrease rubidium influx in monocyte preparations obtained from human blood. Only the ouabain-sensitive portion of rubidium influx was inhibited. The action of elastin peptides is strongly concentration-dependent; the maximal activity observed in the above reactions was less than 1 microgram/ml. These results suggest that elastin peptides may play a role in the regulation of the biological activity of mesenchymal cells, in the proximity of which they are released by the action of elastase-type enzymes. Such enzymes were demonstrated in aorta smooth muscle cells (membrane-bound serine protease) and in fibroblasts (metalloprotease). Monocytes and polymorphonuclear leukocytes were also shown to carry elastase-type enzymes. The release of peptides from elastin by elastase-type enzymes and the action of such peptides on the ion fluxes through the cell membrane may well be involved in mechanisms of the modulation of the phenotype of mesenchymal cells during aging as well as in the development of age-dependent pathologies such as arterioclerosis.

Adult↗
PubMed1987

Increased biosynthesis and processing of fibronectin in fibroblasts from diabetic mice.

Diabetic connective tissues exhibit a deranged regulation of extracellular matrix biosynthesis. Fibronectin is shown to be increased in human dermal connective tissue by immunofluorescence, mainly at the dermoepidermal and capillary basement membranes. The rate of fibronectin biosynthesis, excretion, and incorporation in a pericellular polymeric form was investigated using genetically diabetic KK mouse skin and fibroblasts as compared to Swiss and C57BL mouse skin and fibroblasts. The rate of incorporation of [35S]methionine into proteins recovered in the culture medium or in deoxycholate and NaDodSO4 or urea extracts was investigated. The rate of incorporation in the medium and deoxycholate extracts was comparable. However, the relative rate of incorporation of the tracer in the NaDodSO4-extractable, pericellular polymeric form was increased in the diabetic KK fibroblasts both for total proteins and for fibronectin. In pulse-chase experiments, the deoxycholate-soluble and NaDodSO4-soluble fractions exhibited a precursor-product relationship. The rate of passage of fibronectin from the deoxycholate-soluble (cellular compartment) form to the NaDodSO4-soluble (pericellular polymeric) form was strongly accelerated in the diabetic fibroblast cultures. These results confirm the increased rate of synthesis of fibronectin in diabetic fibroblasts as well as its processing from the cellular compartment to the polymeric pericellular form. The increase of fibronectin in diabetic connective tissues, in the matrix as well as in the basement membranes, may play a role in the mechanism of micro- and macroangiopathies and in the perturbed permeability characteristics of the diabetic capillaries, and as a glycoprotein it may contribute to the increased periodic acid/Schiff reagent staining of diabetic capillary basement membranes.

Animals↗
PubMed1987

Hybridoma antibodies to the lipid-binding site(s) in the amino-terminal region of fibronectin inhibits binding of streptococcal lipoteichoic acid.

In this report, we present evidence to suggest that streptococci and lipoteichoic acid (LTA) interact with a fatty acid binding site located near the NH2-terminus of fibronectin. The evidence is based on the following observations. Antibodies directed against a synthetic peptide (residues 1-30 of the amino-terminus of fibronectin) reacted with the two thermolysin-generated peptides (24 and 28 kilodaltons [kDa]) that were adsorbed by and eluted from streptococci. The adsorption of the 24- and 28-kDa peptides to streptococci was inhibited by LTA. The two monoclonal antibodies that inhibited the binding of LTA to fibronectin reacted only with the 24- and 28-kDa fragments of fibronectin. Conversely, LTA, as well as lauric acid and oleic acid, blocked the binding of the same monoclonal antibodies to fibronectin. LTA had no effect on the binding of hybridoma antibodies directed against the collagen or cell-binding domain.

Animals↗
PubMed1986

Effect of elastin peptides on human monocytes: Ca2+ mobilization, stimulation of respiratory burst and enzyme secretion.

The effect of elastin peptides (Kappa-elastin) was investigated on human monocytes. The data presented here indicate that elastin peptides increase the intracellular Ca2+ level measured by Quin 2 fluorescence and mediate the release of beta glucuronidase and elastase. The O2 consumption and H2O2 release were stimulated in a dose-dependent manner. The early rise of cAMP was followed by a return to the original level at 30 min and by a concomitant increase of cGMP level. The action of elastin peptides on intracellular calcium level and cGMP levels may well be related to its previously demonstrated chemotactic activity. These activities may well play a role in the modifications of the extracellular matrix following elastin degradation as observed in atherosclerosis, emphysema and aging.

Calcium↗
PubMed1986

Proteoglycans from pig aorta. Comparative study of their interactions with lipoproteins.

Different proteoglycans (PGs) were isolated from pig aorta for aggregation studies with hyaluronic acid and human low-density lipoproteins (LDL). Extraction of the intima-media with 4M-guanidinium chloride and digestion of the residue with collagenase solubilized 91% of aortic hexuronic acid content. From the guanidinium chloride extract two PGs were isolated by ion-exchange and gel-permeation chromatography: proteochondroitin sulphate (PGI) with a protein-core apparent Mr of 250 000 and proteodermatan-chondroitin sulphate (PGII) with a protein-core apparent Mr of 55 000. Only PGI forms high-Mr aggregates with hyaluronic acid. From the collagenase digest two other PGs were isolated: proteoheparan sulphate and proteochondroitin sulphate (PGIII and PGIV respectively). PGIV had a smaller hydrodynamic size than PGI. PGI and PGII formed insoluble complexes with human LDL in the presence of Ca2+. PGIII or PGIV did not form precipitates with the LDL. PGI and PGII, but neither PGIII nor PGIV, were bound to LDL-Sepharose. The main peaks of PGI and PGII were eluted from LDL-Sepharose with 60 mM- and 90 mM-NaCl respectively. The results indicate that aortic PGs have different interacting potentials with lipoproteins, depending on their Mr and their glycosaminoglycan composition.

Animals↗
PubMed1986

Inducible adhesion of mesenchymal cells to elastic fibers: elastonectin.

The addition of highly purified elastic fibers to confluent human skin fibroblast or porcine aorta smooth muscle cell cultures resulted in a time-dependent, strong adhesion of the fibrils to the cell surface. The kinetics of adhesion was studied by video/time-lapse cinematography. After a 0.5-1 hr lag period, adhesion progressed to a maximum amount in 3-6 hr in the described conditions. Adhesion is strongly accelerated by the prior addition of soluble elastin peptides (kappa-elastin) to the cultures. Cycloheximide inhibits this induced adhesion. Adherent elastic fibers can be detached by treatment with elastase and trypsin but not with collagenase. The radioactive proteins adhering to elastic fibers, after a 6-hr incubation of the induced cultures in presence of [35S]methionine, were extracted and analyzed by NaDodSO4/PAGE. The proteins strongly adhering to the elastic fibers had apparent molecular sizes of about 120, 67, 60, and 45 kDa. Only the 120-kDa protein band showed a significant increase of its associated radioactivity in the induced cultures as compared to the noninduced cultures. We propose that the 120-kDa protein is responsible for the induced adhesion of mesenchymal cells to elastic fibers and designate it "elastonectin."

Animals↗
PubMed1986

Physicochemical and ultrastructural properties of cholesterol esters bound to elastin.

Physicochemical and ultrastructural properties of cholesterol ester complexes of fibrous elastin and of K-elastin were studied using differential scanning calorimetry (DSC) and electron-microscopy. The number of molecules of the different fatty acids retained in these elastin complexes varied between large limits according to the nature of the fatty acid of the cholesterol ester, ranging from 0.1 microgram/200 mg of elastin for cholesterol arachidonate to 48 micrograms/200 mg elastin for cholesterol palmitate. The ultrastructural studies confirmed the association of cholesterol esters with fibrous elastin and soluble K-elastin. The DSC-data showed that the temperature of transition between the crystalline and the liquid crystalline state shifts towards higher temperatures (well above body temperature) when cholesterol esters are bound to elastin: no liquid crystalline mesophase was observed in cholesterol oleate or linoleate-elastin complexes at 37 degrees C; melting of the crystalline structures took place at 51 degrees C and at 42 degrees C for the cholester Holeate and linoleate-elastin complexes respectively. These results indicate that in the elastin-bound form the crystalline-liquid crystalline transition is inhibited. The "stabilization" of the crystalline structure of cholesterol esters by fibrous elastin may be of biological significance and account for the crystalline deposits seen in advanced atherosclerotic lesions at sites of elastic fiber breakdown.

Calorimetry, Differential Scanning↗
PubMed1986

Study of elastase-type activity in blister fluids of recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized clinically by blister formation due to minor trauma and ultrastructurally by a progressive disappearance of anchoring fibrils at the dermoepidermal junction and of the oxytalan-type fibers which belong to the elastic fiber system. In this study, we determined the elastase-type activity in blister fluid obtained from 8 patients suffering from RDEB as compared to the suction fluid of experimental blisters in a healthy person and to the blister fluid of a patient suffering from epidermolysis bullosa simplex. One patient with dominant dystrophic epidermolysis of the albopapuloid type was also studied. Seven of the eight children with RDEB showed highly elevated values. The eighth child, treated with etretinate, as well as the patient suffering from dominant epidermolysis bullosa had moderately increased values. The determination of elastase-type activity in the blister fluid could therefore be useful to establish the differential diagnosis of recessive dystrophic epidermolysis bullosa.

Blister↗
PubMed1986

Increased type-III/type-I collagen ratios in diabetic human conjunctival biopsies.

The ratio of type-III to type-I collagen is measured in human conjunctival biopsies from control and diabetic subjects. The tissue is digested by CNBr and the resulting peptides are quantified by SDS polyacrylamide gel electrophoresis. The peptides used are alpha 1-(I)CB7 and alpha 1-(III)CB8. In control population, of type-III collagen slightly increases with age. In two diabetic populations, (juvenile onset diabetes and maturity onset diabetes), the percentage of type-III collagen is significantly higher than in age-matched control groups. These data plus those previously obtained on genetically diabetic mice indicate that diabetes mellitus affects the expression of interstitial collagen phenotype. Preliminary results on prediabetic subjects suggest the role of genetic factors in such alterations.

Adult↗
PubMed1986

[Macromolecules of the extracellular matrix].

In this introductory paper, the authors review the macromolecular basis of extracellular matrix. 4 principal groups of macromolecules are known: collagen, elastin, proteoglycans and structural glycoproteins. The phylogenetic evolution lead to the great complexity of this matrix. The interest of a more specific pharmacology directed towards this extracellular matrix is emphasised.

Chemical Phenomena↗
PubMed1985

Selective decrease of type I collagen synthesis in Fraser mice skin.

Quantification and biosynthesis of type I and type III collagens were determined in skin of control and Fraser mice (CatFraser mutation), which exhibit a genetically determined cataract. Skin organ cultures were labelled with [3H]proline. Pepsin-solubilized collagens were studied using three different approaches: (a) differential salt precipitation at neutral pH, followed by SDS-polyacrylamide gel electrophoresis; (b) differential salt precipitation at acid pH followed by SDS-polyacrylamide gel electrophoresis. (c) CNBr peptide analysis. These methods gave consistent and reproducible results, indicating a selective decrease of type I collagen in Fraser mouse skin as compared to control mouse skin. Metabolic labelling of skin organ cultures showed a decreased specific radioactivity of hydroxy[3H]proline in type I collagen of Fraser mouse skin. The concordant results of these experiments suggest a genetically determined alteration of interstitial collagen metabolism in the Fraser mutation apparently specifically concerning the expression of type I collagen gene(s).

Animals↗
PubMed1985

Fatty acid peptide derivatives as model compounds to protect elastin against degradation by elastases.

Peptide sequences which fit the extended binding sites of porcine pancreatic elastase and human leukocyte elastase were covalently coupled to oleic acid. These compounds behave as competitive inhibitors towards both elastases. The coupling of fatty acid moiety to the peptide greatly decreases its inhibitor constant (Ki) vs human leukocyte elastase (Ki for Oleoyl(Ala)2ProValine: 3.0 (10(-6)M). It is less active on porcine pancreatic elastase (Ki for Oleoyl(Ala)2ProAlanine: 3.8 10(-4)M). The modifications of the carboxylic end group of the peptide to an aldehyde further greatly enhanced the inhibition capacity of the compound towards leukocyte elastase (Ki for Oleoyl(Ala)2ProAlaninal: 0.7 microM). Oleoyl peptide derivatives were seen to bind in a saturable fashion to purified insoluble elastin, and decreased the susceptibility of the macromolecule to hydrolysis by both pancreatic and leukocyte elastases. As stoichiometric quantities of elastase (vs inhibitor) could not desorb 3H-oleoyl(Ala)2Pro-Val bound to insoluble elastin, it is postulated that oleoyl peptide derivatives may act as bifunctional agents. This contention was further strengthened by the comparison of the adsorption curves of elastase to untreated insoluble elastin and elastin saturated with oleoyl peptide derivatives respectively. It was shown finally that Oleoyl(Ala)2Pro-Valine was also capable of inhibiting elastases in their adsorbed form to insoluble elastin.

Adsorption↗
PubMed1985

Biosynthesis of hyaluronic acid, heparan sulfate and structural glycoproteins in hamster lung explants during elastase induced emphysema.

Hyaluronic acid (HA), heparan sulfate (HS) and structural glycoproteins (SGP) were investigated in explant cultures of hamster lungs by studying incorporation of 14C-glucosamine (14C GlcN) on the first and on the 24th day after intratracheal administration of pancreatic elastase. The different 14C radiolabeled macromolecules were extracted sequentially by 0.4 M guanidinium chloride (0.4 M GUA), 4 M GUA and collagenase digestion. At one day following elastase injury, a 4.2 fold increase of 14C GlcN incorporation into HA released in 0.4 M GUA extract and a 2.6 fold increase into HS released in the collagenase digests were observed compared to control tissues; at 24 days, the increased 14C GlcN incorporation into HA and HS persist but to a lesser extent. Polyacrylamide gel electrophoresis and isoelectric focusing carried out on 4 M GUA extracts, demonstrated identical quantitative and qualitative distribution of 14C GlcN between the major SGP (140 and 110 K with pI 7.8 and 4.5 respectively) in the normal and the experimental groups. These results indicate that pulmonary SGP biosynthesis is not modified at one and 24 days after elastase injury, whereas HA and HS biosynthesis are consistently increased. These results suggest a specific role of these macromolecules in emphysematous injury of the lung.

Animals↗