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C Favreau

Publications and source records attributed to C Favreau.

16 recordsLinked to original sources
PubMed2001

Biochemical characterization of nuclear pore complex protein gp210 oligomers.

The membrane-spanning glycoprotein gp210 is a major component of the nuclear pore complex. This nucleoporin contains a large cisternal N-terminal domain, a short C-terminal cytoplasmic tail, and a single transmembrane segment. We show here that dimers of native gp210 can be isolated from cell extracts by immunoprecipitation, and from purified rat liver nuclear envelopes by velocity sedimentation and gel filtration. Cross-linking of proteins in isolated membranes prior to solubilization dramatically increases the proportion of dimers. The dimers are SDS-resistant, as previously observed for some integral membrane proteins of cis-Golgi and plasma membrane proteins, including glycophorin A. Larger oligomers of gp210 can also be obtained by gel filtration and denaturing electrophoresis, but unlike the dimers are dissociated by reduction and heating in the presence of SDS. We propose that gp210 is organized into the pore membrane as a large array of gp210 dimers that may constitute a luminal submembranous protein skeleton.

Animals↗
PubMed1996

Cell cycle-dependent phosphorylation of nucleoporins and nuclear pore membrane protein Gp210.

During mitosis in higher eukaryotic cells, the nuclear envelope membranes break down into distinct populations of vesicles and the proteins of the nuclear lamina and the nuclear pore complexes disperse in the cytoplasm. Since phosphorylation can alter protein-protein interactions and membrane traffic, we have examined the cell cycle-dependent phosphorylation of nuclear pore complex proteins. Nonmembrane nucleoporins Nup153, Nup214, and Nup358 that are modified by O-linked N-acetylglucosamine and recognized by a monoclonal antibody were phosphorylated throughout the cell cycle and hyperphosphorylated during M phase. Pore membrane glycoprotein gp210, that has a cytoplasmic, carboxyl-terminal domain facing the pore, was not phosphorylated in interphase but specifically phosphorylated in mitosis. Mutant and wild-type fusion proteins containing the cytoplasmic domain of gp210 were phosphorylated in vitro and their phosphopeptide maps compared to that of mitotic gp210. This analysis showed that Ser1880 of gp210 was phosphorylated in mitosis, possibly by cyclin B-p34cdc2 or a related kinase. Several nuclear pore complex proteins are therefore differentially phosphorylated during mitosis when pore complexes disassemble and reassemble.

Acetylglucosamine↗
PubMed1995

Role of monocyte/macrophage derived matrix-metalloproteinases (gelatinases) in prolonged skin inflammation.

Neutral metalloproteinase activities in dermal extracellular space have been studied in several models of prolonged cutaneous inflammation in guinea pigs, by the following techniques: lysis of type I 14C-collagen fibrils, electrophoretic analysis of types I or IV collagen hydrolytic fragments and zymography. For 2-3 weeks, in parallel to mononuclear cell infiltration, collagenase activity was increased 3-4-fold. Constitutive gelatinases (67 and 72 kDa) augmented and larger molecular species emerged (92, 110 and 185 kDa), all of neutral metalloproteinase type. Guinea pig peritoneal monocytes/macrophages cultured with appropriate stimulation released large gelatinases in a similar set (92, 110, 210 kDa). These were purified from culture media by gelatin affinity and used in vivo as follows: (a) direct injection of monocyte/macrophage gelatinases; (b) injection of collagen I fragments (M(r) < 10,000) split off by gelatinases from preincubated (pH 5 and 38.5 degrees C) collagen I. In both instances, a mononuclear cell invasion of dermis occurred, indistinguishable from prolonged inflammation. These analogies suggest that monocyte/macrophage-derived metalloproteinases have an early and basic participation in the mechanism of prolonged inflammation.

Animals↗
PubMed1992

Protection of vascular basement membrane and microcirculation from elastase-induced damage with a fluorinated beta-lactam derivative.

N-(2-chloromethylphenyl) 3,3-difluoroazetidin-2-one (AA 231-1), a specific suicide-type inhibitor of elastase which is known to suppress the lysis of chromogenic oligopeptides, elastin and elastic fibers, is effective also in preventing the degradation of the vascular basement membrane. The degradation of porcine glomerular basement membrane by purified human leukocyte elastase (HLE), was reduced in proportion of inhibitor dose (8.3 microM for 50% inhibition). It is noteworthy that there was no reduction of the inhibitory effect when the addition of AA 231-1 was delayed for 1 h after the addition of the enzyme to the substrate. In the guinea pig, reduction of the dermal microhemorrhage due to HLE was related to the dose of inhibitor and to its preincubation time with HLE before intradermal injection. The inflammatory hemorrhage associated with the Arthus skin reaction was moderately depressed by AA 231-1 in situ. A part of the vascular permeability induced by HLE also responded to the inhibitor. In spite of the tissular diffusion and the time-dependence parameters which restrict responsiveness of elastase to AA 231-1 in vivo this biochemical compound should be helpful in the study and possibly the cure of vascular injury related to elastase.

Amino Acid Sequence↗
PubMed1992

Prevention of some types of inflammatory damage using AA 231-1, a fluorinated beta-lactam.

The leukocyte elastase inhibitory activity of AA 231-1, a suicide substrate, was investigated in the presence of elastin, a natural substrate of elastase, and its efficiency to reduce the degradation of basement membrane and haemorrhage induced by elastase was analysed. Elastin only moderately decreased the inhibitory efficiency of AA 231-1. The digestion by human leukocyte elastase (HLE) of glomerular basement membrane prepared from pig kidney was prevented in the presence of AA 231-1. Intradermal microvascular haemorrhage was also significantly inhibited by AA 231-1. These results suggest that AA 231-1 may be a valuable candidate as an anti-inflammatory agent.

Animals↗
PubMed1991

A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator.

c[Arg-aB-(CH2+SCH3 phi)-Gly4] was designed and studied as a mechanism-based inactivator (suicide substrate) for plasminogen activators (u-PA and t-PA) and plasmin. This compound inhibited u-PA and fulfills criteria expected for the involvement of an enzyme-activated inhibitor: first-order and irreversible process, saturation kinetics, protection by substrate. The limiting first-order rate constant kinact and the apparent enzyme-inhibitor dissociation constant KI were 0.021 s-1 and 9 microM, respectively at pH 7.5 and 25 degrees C. The activation of plasminogen by u-PA is compromised after this enzyme has been treated by the reagent. Plasmin and t-PA were inactivated 40- and 2330-fold less efficiently than u-PA, respectively.

Amino Acid Sequence↗
PubMed1990

Reaction of thrombin and proteinases of the fibrinolytic system with a mechanism-based inhibitor, 3,4-dihydro-3-benzyl-6-chloromethylcoumarin.

Thrombin, plasmin and tissue plasminogen activator (one- and two-chain forms) were examined with respect to their reaction with the suicide substrate, 3,4-dihydro-3-benzyl-6-chloromethylcoumarin, at 4 degrees C. The enzymes were irreversibly inhibited and the apparent second-order rate constants ki/Ki were 31,000, 316, 187 and 250 M-1.s-1, respectively. The extent of fibrin clot lysis induced by urokinase and two-chain tissue plasminogen activator was considerably decreased after treatment of these enzymes with the dihydrocoumarin derivative (molar excess of inhibitor over enzyme ranging from 6 to 21 for urokinase and 50 to 1500 for tissue plasminogen activator). This inhibitor has been tested as anticoagulant in human plasma and was effective at prolonging the prothrombin time from 12 to 40 s.

Blood Coagulation↗
PubMed1990

Biological evaluation of the inhibition of neutrophil elastase by a synthetic beta-lactam derivative.

A novel beta-lactam derivative, N-(2-chloromethylphenyl) 3,3-difluoroazetidin-2-one, which behaves as a time-dependent inactivator of leukocyte elastase, has been tested in biological models designed to detect its potential therapeutic value in the treatment of emphysema. Its effect on two types of leukocyte elastase, purified human leukocyte elastase and elastase freshly discharged upon stimulation of guinea pig polymorphonuclear neutrophils, was examined using three methods: the cleavage of a chromogenic peptide substrate, MeO-Suc-Ala-Ala-Pro-Val-NA, the lysis and solubilization of tritiated elastin and the microscopic examination of the damage to lung elastic network. The inhibitor was shown to be effective at preventing proteolysis due to leukocyte elastase. Besides its low cellular toxicity, no apparent hindrance of its efficiency was found in the above quasi in vivo environment. This suggests that this inhibitor may be of potential therapeutic value in elastase-related pathology.

Amino Acid Sequence↗
PubMed1984

Mechanisms of fatty acid effects on sarcoplasmic reticulum. III. The effects of palmitic and oleic acids on sarcoplasmic reticulum function--a model for fatty acid membrane interactions.

The mechanism by which palmitic and oleic acids modify calcium sequestration by sarcoplasmic reticulum vesicles was investigated by examining the effects of these fatty acids on calcium-dependent ATPase activity, on the phosphoenzyme intermediates found during calcium sequestration reactions, and on passive membrane permeability to calcium. The calcium sequestered in the presence of these fatty acids was also characterized by determining the amount exchangeable with the extravesicular pool or released by the ionophore A23187. In the presence of 50 microM ATP, 18 microM palmitic acid enhanced and 18 microM oleic acid inhibited calcium sequestration, whereas both fatty acids stimulated ATPase activity. Neither fatty acid had significant effects on the amount or distribution of the phosphoenzyme formed during the calcium transport reaction. Palmitic acid stimulated calcium sequestration only when ATP was present. Oleic acid caused the release of a portion of the accumulated calcium during ATP-supported calcium sequestration and also enhanced the release observed in ATP-depleted reactions. A portion of the calcium sequestered in the presence of palmitic acid appears to be incorporated into a nonexchangeable and ionophore-insensitive calcium pool, although the latter was estimated to be considerably larger than the nonexchangeable pool. These data support the hypothesis that oleic acid inhibits calcium sequestration by increasing membrane permeability to calcium, whereas palmitic acid appears to stimulate calcium sequestration by interacting with a portion of the calcium within the vesicles to form a separate, poorly exchangeable calcium pool.

Adenosine Triphosphate↗
PubMed1984

Mechanisms of fatty acid effects on sarcoplasmic reticulum. II. Structural changes induced by oleic and palmitic acids.

The interaction of micromolar concentrations of palmitic and oleic acids with the sarcoplasmic reticulum membrane was studied by electron microscopic techniques in an attempt to define their different effects on ATP-induced calcium sequestration in sarcoplasmic reticulum vesicles. Oleic acid had a concentration-dependent effect on the morphology of sarcoplasmic reticulum vesicles, promoting vesicle fusion and eventual solubilization. Palmitic acid did not alter the morphology of sarcoplasmic reticulum, but its probable site(s) of interaction could be determined. In the presence of palmitic acid, large lamellar structures that formed external to sarcoplasmic reticulum vesicles are probably composed of pure palmitic acid and/or palmitic acid/phospholipid mixed "micelles," but internalization of palmitic acid into sarcoplasmic reticulum vesicles was not detected. Palmitic acid reduced the phospholipid content of sarcoplasmic reticulum membranes with a preservation of the average interparticle protein spacing as observed in freeze-fracture electron micrographs. Thus, palmitic acid appears to be incorporated into the sarcoplasmic reticulum lipid bi-layer. Oleic acid inhibition of ATP-induced calcium sequestration by sarcoplasmic reticulum vesicles is probably caused by net permeability changes of the membrane. A structural mechanism for palmitic acid stimulation of ATP-induced calcium sequestration is proposed in light of the probable insertion of palmitic acid into the sarcoplasmic reticulum lipid bilayer.

Adenosine Triphosphate↗
PubMed1983

Release of a lymphokine-like plasminogen activator by stimulated B lymphocytes.

This report extends to the guinea pig the discovery of a lymphocyte plasminogen activator (LPA) previously described in the mouse. In the guinea pig, we have identified enzymatic activity similar to that in the mouse, but which has two distinct components: first, as in the mouse, a membrane-bound molecule present even in the quiescent lymphocyte (mLPA), and second, a previously unreported soluble molecule appearing in the culture medium after appropriate cell stimulation (sLPA). This sLPA, like most known lymphokines, was released by in vitro recall of sensitized lymphocytes by the antigen and by direct contact with a mitogen. There was a parallel evolution in the culture for sLPA and for some other well-known lymphokines (LT, LIF), and their detection thresholds were of the same order. A possible activation of plasminogen (Pg) by macrophages contaminating the lymphocyte cultures was carefully rule out. sLPA was produced by lymph node lymphocytes as well as by blood lymphocytes, but not by spleen cells, thymocytes, or peritoneal lymphocytes. A study of the kinetics of the release of sLPA, together with that of metabolic modifiers, suggested that an intracellular synthesis precedes the secretion of the molecule. Data obtained from the use of B- and T-enriched subpopulations or B- or T-dependent antigens and mitogens point to the B lymphocytes as the major, if not exclusive, source of sLPA. The choice of synthetic chromogenic substrates S 2251, S 2444, and S 2288 in some experiments led us to confirm most of the above properties of sLPA with still greater precision and reliability.

Animals↗
PubMed1981

Plasminogen activation by normal B lymphocytes, a function associated with the cell membrane.

When purified murine plasminogen was added to cultures of mouse spleen B cells, active plasmin progressively appeared in the supernatants. This reaction, resulting from the specific cleavage of the plasminogen by lymphocyte plasminogen activator (LPA), was measured in a fibrinolysis assay using 125I-fibrinogen. T cells were totally ineffective; under certain conditions, they could even antagonize the B cell action. Of the various B populations studied, i.e., obtained from spleen, lymph nodes, or blood of various mouse strains, all expressed the same property of plasminogen activation, which concerned mainly medium-sized B cells. Since only slight activities were detected in extracellular or intracellular compartments, a membrane-associated proteolytic enzyme may be responsible for plasminogen activation. Submitted to a series of group-specific antiproteases, the lymphocyte plasminogen activator essentially behaved as a serine-protease, with sensitivity to diisopropyl fluorophosphate, phenyl methyl sulfonyl fluoride, and nitro phenyl guanidino benzoate. The fast renewal of the enzyme in the membrane was also demonstrated by different techniques, using modifiers of cell physiology, like cycloheximide and dexamethasone, or following the reexpression of the enzyme by the cell kinetically.

Animals↗
PubMed1975

Specific and nonspecific channels leading to proteolysis in antibody preparations.

Current ways of fractionating immune sera give rise in the guinea-pig species to an active plasmin that contaminates antibody preparations. The enzyme appears as a free molecule separable from the immunoglobulin. In contrast, when a purified antibody has been obtained through the immunoabsorption process, plasmin activated during the contact between antibody and polymer binds firmly to the immunoglobulin. This phenomenon occurs separately and indistinctly with IgG1 and IgG2. Moreover, the alternate pathway of complement activation could be a tributary to the plasmin system.

Animals↗