Biomedical subjects
Publications and source records attributed to M Gaudry.
Electron-lattice energy exchanges are investigated in gold and silver nanoparticles with sizes ranging from 30 to 2.2 nm embedded in different environments. Femtosecond pump-probe experiments performed in the low-perturbation regime demonstrate a strong increase of the intrinsic electron-phonon interaction for nanoparticles smaller than 10 nm due to a confinement effect.
OBJECTIVE: We assessed the clinical and histologic features of angiogenesis inhibition in a transgenic mouse model of arthritis that closely resembles rheumatoid arthritis (RA) in humans. METHODS: KRN/NOD mice, which spontaneously develop arthritis, were treated with TNP-470, an angiogenesis inhibitor. Disease was monitored by use of clinical indices and histologic examinations; circulating blood levels of vascular endothelial growth factor were determined by enzyme-linked immunosorbent assay. RESULTS: In the preventive protocol, with TNP-470 administration at a dosage of 60 mg/kg of body weight, the onset of arthritis was delayed and its clinical intensity was rather mild; 100% of placebo-treated transgenic mice developed arthritis that led to severe articular destruction. At a dosage of 90 mg/kg of TNP-470, the appearance of clinical signs was delayed for a longer period of time and disease was almost abolished. The therapeutic regimen alleviated clinical signs only when given during the very early stage of disease. Reductions in cartilage and bone destruction by TNP-470 treatment were observed histologically, a feature that was still evident at 30 and 80 days after injections were withdrawn. CONCLUSION: Our demonstration that in vivo administration of an angiogenesis inhibitor suppresses arthritis and protects from bone destruction provides new insight into the pathogenesis of the disease and opens new possibilities in the treatment of RA in humans.
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Neutrophil superoxide production can be potentiated by prior exposure to "priming" agents such as granulocyte/macrophage colony stimulating factor (GM-CSF). Because the mechanism underlying GM-CSF-dependent priming is not understood, we investigated the effects of GM-CSF on the phosphorylation of the cytosolic NADPH oxidase components p47(phox) and p67(phox). Preincubation of neutrophils with GM-CSF alone increased the phosphorylation of p47(phox) but not that of p67(phox). Addition of formyl-methionyl-leucyl-phenylalanine (fMLP) to GM-CSF-pretreated neutrophils resulted in more intense phosphorylation of p47(phox) than with GM-CSF alone and fMLP alone. GM-CSF-induced p47(phox) phosphorylation was time- and concentration-dependent and ran parallel to the priming effect of GM-CSF on superoxide production. Two-dimensional tryptic peptide mapping of p47(phox) showed that GM-CSF induced phosphorylation of one major peptide. fMLP alone induced phosphorylation of several peptides, an effect enhanced by GM-CSF pretreatment. In contrast to fMLP and phorbol 12-myristate 13-acetate, GM-CSF-induced phosphorylation of p47(phox) was not inhibited by the protein kinase C inhibitor GF109203X. The protein-tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitor wortmannin inhibited the phosphorylation of p47(phox) induced by GM-CSF and by fMLP but not that induced by phorbol 12-myristate 13-acetate. GM-CSF alone did not induce p47(phox) or p67(phox) translocation to the membrane, but neutrophils treated consecutively with GM-CSF and fMLP showed an increase (compared with fMLP alone) in membrane translocation of p47(phox) and p67(phox). Taken together, these results show that the priming action of GM-CSF on the neutrophil respiratory burst involves partial phosphorylation of p47(phox) on specific serines and suggest the involvement of a priming pathway regulated by protein-tyrosine kinase and phosphatidylinositol 3-kinase.
Vascular endothelial growth factor (VEGF ), an endothelial cell mitogen, is a potent angiogenic factor produced by several cell types. Whether human neutrophils are potential producers of VEGF has not yet been described. The present work shows that phorbol-12-myristate 13-acetate (PMA), fMet-Leu-Phe, and tumor necrosis factor-alpha (TNF-alpha) triggered a time-dependent secretion of VEGF by human neutrophils. Cells incubated with 50 ng/mL of PMA released significant amounts of VEGF after 15 minutes. Because the extracellular content of VEGF in human neutrophils supernatants remained constant over a period of 2 to 24 hours and because PMA is a potent inducer of human neutrophil degranulation, the PMA-induced secretion of VEGF may be due to a pre-existing intracellular pool of this molecule. This hypothesis was reinforced by the absence of cycloheximide effect on the PMA-induced secretion of VEGF. The existence of an intracellular pool of VEGF was confirmed by measuring the intracellular content of VEGF in resting neutrophils. A dosedependent inhibition of PMA-induced VEGF secretion was observed when the cells were incubated in the presence of pentoxifylline, a methylxanthine known to inhibit neutrophil degranulation. To confirm the implication of neutrophil degranulation in VEGF release, the effects of two inducers of physiologic degranulation, fMet-Leu-Phe and TNF-alpha, were determined. Both agonists induced a release of VEGF in the absence of cytochalasin B, confirming the involvement of neutrophil degranulation and suggesting the intracellular localization of VEGF in the specific granule fraction. In addition, the kinetics of fMet-Leu-Phe- and TNF-alpha-induced secretion of lactoferrin were similar to those of VEGF release induced by these two both agonists. The subcellular fractionation of human neutrophils showed a granule-specific distribution of the intracellular pool of VEGF in resting neutrophils. The finding that human neutrophils contain an intracellular pool of VEGF, secreted in the extracellular space under PMA-, fMet-Leu-Phe-, and TNF-alpha-induced degranulation, suggests a role for human neutrophils as cellular effectors of physiologic as well as pathologic angiogenesis.
In order to localize the active site of the vitamin K-dependent carboxylase, we developed an affinity probe containing the propeptide and the first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino-terminal and hydrophilic carboxy-terminal domains of the carboxylase, in contrast with previous work which localized both the catalytic and the propeptide binding site within the amino-terminal hydrophobic domain. Amino acid analysis revealed that the mass of an amino-terminal fragment is seriously underestimated by SDS-PAGE. Reanalysis of the published data in light of this information suggests that a portion of the propeptide binding site resides within the carboxy-terminal hydrophilic domain.
The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O-2) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67(phox). In this study, we found that activation of human neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67(phox). Using an anti-p67(phox) antibody or an anti-p47(phox) antibody, we showed that phosphorylated p67(phox) and p47(phox) form a complex. Phosphoamino acid analysis of the phosphorylated p67(phox) revealed only 32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67(phox) is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67(phox) in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67(phox) was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67(phox) is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.
Activation of various cell types by different agonists is known to stimulate a transient increase in the level of tyrosine phosphorylation of certain cellular proteins. Such phosphorylation is essential for mediating signalling by these agonists. The preservation of the tyrosine phosphorylation of proteins in lysates has proven to be a difficult task in neutrophils because of their large arsenal of proteases and phosphatases. Here we describe a technique that we found useful for preserving the tyrosine phosphorylation of cellular proteins. The technique depends on the denaturing lysis of neutrophils followed by the removal of the denaturing agents using Sephadex columns. Preparing neutrophil lysates by this technique has proven to be reliable in terms of maintaining the stability of the tyrosine phosphorylated proteins of various molecular weights and their subsequent immunoprecipitation and identification.
Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists. The effects of colchicine were shared by other anti-microtubule agents (nocodazole and vinblastine) but not by its inactive analogue beta-lumicolchicine, trimethylcolchicinic acid, indomethacin, or phenylbutazone. Furthermore, the (enhancing as well as inhibitory) effects of colchicine on tyrosine phosphorylation and superoxide anion production were reversed by taxol. Finally, in human cytoplasts colchicine again inhibited microcrystal-stimulated tyrosine phosphorylation but did not change chemotactic factor-stimulated phosphorylation. These data strongly support the hypothesis that microtubule-related mechanisms are involved in the modulation of the tyrosine phosphorylation response in human neutrophils, and suggest that a relationship may exist between the augmentation of tyrosine phosphorylation and of the stimulation of the NADPH oxidase induced by chemotactic factors.
The functional responsiveness of human neutrophils is known to be initiated and modulated by protein tyrosine phosphorylation. The regulation of the levels of tyrosine phosphorylation is most likely the result of the coordinated actions of tyrosine kinases and phosphatases, which have so far been only very partially characterized. In the present study, we present evidence demonstrating that the stimulation of neutrophils by a variety of agonists (soluble as well as particulate) leads to the activation of the src-related tyrosine kinase lyn. The stimulation of tyrosine kinase activity of lyn was detected using an immune kinase assay as well as an in situ labeling technique. Phosphoaminoacid analysis of lyn indicated that the autophosphorylation of the kinase was exclusively on tyrosine residues. The time course of the activation of lyn is consistent with its playing a role in the early tyrosine phosphorylation responses of neutrophils. The ability of agonists with widely varying functional end responses to stimulate the activity of lyn indicates that this event plays a key and central role in the control of the activation of human neutrophils.
Using both primary- and tertiary-structure comparisons, we have established new structural similarities shared by reductases, epimerases and dehydrogenases not previously known to be related. Despite the low sequence identity (down to 10%), short consensus segments are identified. We show that the sequence, the active site and the supersecondary structure are well conserved in these proteins. New homologues (the protochlorophyllide reductases) are detected, and we define a new superfamily composed of single-domain dinucleotide-binding enzymes. Rules for the cofactor-binding specificity are deduced from our sequence alignment. The involvement of some amino acids in catalysis is discussed. Comparison with two-domain dehydrogenases allows us to distinguish two general mechanisms of divergent evolution.
During the biosynthesis of fungal melanin, tetrahydroxynaphthalene reductase catalyzes the NADPH-dependent reduction of 1,3,6,8-tetrahydroxynaphthalene (T4HN) into (+)-scytalone and 1,3,8-trihydroxynaphthalene into (-)-vermelone. The enzyme from Magnaporthe grisea, the fungus responsible for rice blast disease, has been purified to homogeneity. It is a tetramer of four identical 30-kDa subunits. A full-length cDNA clone of about 1 kb encoding T4HN reductase has been isolated from a cDNA library constructed in the lambda ZAP II vector and characterized. The clone contains a 846-bp open reading frame. Translation of the DNA sequence gave a 282-residue amino acid sequence with a calculated molecular mass of 29.9 kDa. Sequences corresponding to the amino-terminal part and three internal proteolytic peptides were present in the translated sequence. T4HN reductase exhibits characteristics of the short-chain alcohol dehydrogenase family. The reductase shares 56% identity with a putative ketoreductase involved in aflatoxin biosynthesis in Aspergillus parasiticus.
Although several tyrosine kinases are present in human neutrophils, little is known regarding the biochemical basis for their activation. We have identified two tyrosine kinase activities in 0.1 and 1% Triton cell extracts of human neutrophils using a non-denaturing gel assay. The first protein tyrosine kinase activity of a faster mobility was associated exclusively with the 0.1% Triton cell extract. The second activity, of slower mobility, was mainly associated with the 0.1% Triton cell extract and to a lesser extent with the 1% Triton cell extract. A modulation of the activities and the distribution of these two tyrosine kinase activities was observed upon stimulation of neutrophils with PDBu (phorbol 12,13-dibutyrate), a direct PKC (protein kinase C) activator. The addition of 1 microM PDBu induced a time-dependent decrease of both tyrosine kinases in the 0.1% Triton cell extract. Although the fast mobility tyrosine kinase activity disappeared completely, the slow mobility tyrosine activity decreased only partially. Concomitantly, an increase in the latter activity was detected in the 1% Triton cell extract. The pattern of tyrosine phosphorylation upon PDBu stimulation was also examined and the results showed that the phorbol ester induced time-dependent increases in the level of phosphotyrosine-containing proteins in at least 10 distinct bands. Two lines of evidence indicated that the effects of PDBu were mediated by PKC: 1) The stereo-isomer of PDBu, 4 alpha-PDBu, did not affect the activities and distribution of the tyrosine kinases, and 2) The PKC inhibitor, RO 318220, prevented the redistribution of the tyrosine kinase activities and inhibited the stimulation of tyrosine phosphorylation induced by PDBu. These results show that the activity and distribution of at least two human neutrophil tyrosine kinases are modulated after the activation of PKC and that the low mobility tyrosine kinase activity is the most sensitive to PDBu. Based on previous studies, the fast mobility tyrosine kinase activity was likely to be a member of the pp60src tyrosine kinase family and the slower one may be related to the pp93fes. Furthermore, these results begin to define the nature of the relationships among the PKC- and the tyrosine kinase-signaling pathways.
The activation of human neutrophils by monosodium urate and calcium pyrophosphate dihydrate crystals is believed to play a critical role in the pathogenesis of arthritides such as acute gout and pseudogout, respectively. In this study, we investigated the potential involvement of tyrosine phosphorylation in microcrystal-mediated activation of human neutrophils. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that triclinic monosodium urate and calcium pyrophosphate dihydrate crystals stimulated a time- and concentration-dependent tyrosine phosphorylation of at least five proteins (pp130, 118, 80, 70, and 60). While phosphoprotein (pp) 118 and pp70 were the major phosphorylated substrates, pp70 was the dominant one in reactivity with antiphosphotyrosine antibodies. When the temporal patterns, as well as the levels of tyrosine phosphorylation for both types of crystals were compared, monosodium urate crystals were found to be more potent activators than calcium pyrophosphate dihydrate crystals. The tyrosine phosphorylation patterns induced by microcrystals differed from those stimulated by other soluble (FMLP, C5a, or leukotriene B4) or particulate (unopsonized latex beads or zymosan) agonists which stimulated preferentially the tyrosine phosphorylation of pp118. The ratio of the intensities of pp118 and pp70 were specific of the stimulation with microcrystals when compared to those observed with the other soluble or particulate agonists. Colchicine, a drug used specifically in the treatment of gout and pseudogout, inhibited microcrystal-induced tyrosine phosphorylation, while beta- and gamma-lumicolchicine were without effect. On the other hand, colchicine failed to inhibit FMLP-induced tyrosine phosphorylation. Furthermore, while colchicine inhibited the activation of the NADPH oxidase by microcrystals, it, on the other hand, enhanced the production of superoxide anions by FMLP. Taken together, these results (a) demonstrate that tyrosine phosphorylation is involved in the mechanism of activation of human neutrophils induced by microcrystals; and (b) suggest, on the basis of the characteristics of the observed patterns of tyrosine phosphorylation, that this response may be specific to the microcrystals and relevant to their phlogistic properties.
We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, potently stimulate a characteristic protein tyrosine phosphorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate agonists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in human blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchicine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-induced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indomethacin and phenylbutazone were similarly without effect on microcrystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyrosine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of microtubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists.
BACKGROUND: Recent studies have indicated that the regulation of the activation of human neutrophils depends on tyrosine phosphorylation and on phospholipase D. Furthermore, a tentative causal relationship between these two signalling pathways has been indirectly implied derived through the use of inhibitors of tyrosine kinases. The fungal metabolite, wortmannin is at present the only compound known to inhibit the receptor-mediated activation of phospholipase D in human neutrophils. Its mechanism of action is presently unknown. EXPERIMENTAL DESIGN: The ability of peripheral blood neutrophils to respond to various agonists with an increase in activity of phospholipase D and an enhancement of tyrosine phosphorylation in the absence or presence of wortmannin was monitored. RESULTS: Wortmannin was found to inhibit the stimulation of tyrosine phosphorylation by fMet-Leu-Phe, and by the inflammatory microcrystals monosodium urate and calcium pyrophosphate dihydrate. This effect of wortmannin was not secondary to inhibition of phospholipase D as U73122, a previously described phospholipase C inhibitor, was also found to inhibit phospholipase D without affecting tyrosine phosphorylation. CONCLUSIONS: The results make it likely that one of the earliest sites of action of wortmannin in human neutrophils is at the level of tyrosine phosphorylation which then exerts a modulatory influence on the activation of phospholipase D.
Erbstatin, a recently described inhibitor of tyrosine kinases, has been used to examine the potential role of tyrosine phosphorylation in human neutrophil locomotion. Preincubation of human neutrophils with erbstatin inhibited both spontaneous and directed migration induced by chemotactic factors such as formylmethionylleucylphenylalanine (fMet-Leu-Phe) and leukotriene B4. The decreased migratory responses were correlated with an inhibition of adherence of neutrophils to serum-coated surfaces. Erbstatin did not, however, affect the adherence of human neutrophils to uncoated surfaces. These results indicated that the inhibitory effects of erbstatin were specific and not due to a generalized alteration of the surface of human neutrophils. To elucidate the mechanism of the inhibitory effect of erbstatin on adherence properties, the expression of the leukocyte integrin Mo1 was studied. Erbstatin induced a small but significant increase in the expression of Mo1, but decreased the stimulation of the expression of Mo1 elicited by fMet-Leu-Phe. These results suggest that mechanisms in addition to alteration of the number of surface integrins are involved in the inhibition of neutrophil adherence and locomotion by erbstatin.
The modulatory influences of phorbol esters on the functional responsiveness of human peripheral blood neutrophils have been investigated. These studies focused on measurements of the levels of cytoplasmic free calcium and of tyrosine phosphorylation as well as on their ability to mount an oxidative response. Short incubation times (< 1 min) with low concentrations of phorbol esters (5-50 nM) were shown to enhance the above indices of neutrophil responsiveness to chemotactic factors such as fMet-Leu-Phe and leukotriene B4. On the other hand, a time- and concentration-dependent inhibition of calcium mobilization and superoxide production was also observed. The effects of the phorbol esters were stereo-specific and were antagonized by a novel protein kinase C inhibitor (RO 318220) but were not affected by the oxidative burst inhibitor diphenyleneiodonium. Pre-incubation of the cells with phorbol 12,13-dibutyrate (PDBu) altered in a concentration-dependent manner the tyrosine phosphorylation pattern stimulated by fMet-Leu-Phe. In addition, the tyrosine kinase inhibitor erbstatin inhibited the priming of the mobilization of calcium induced by PDBu. These data demonstrate the rapidity of the effects of the activation of protein kinase C, their potential to modulate positively the early events of the excitation-response coupling sequence and the complexity of the functional interrelationships among the various cellular activation pathways available to human neutrophils and other non-muscle cells.