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Biomedical subjects

S Paris

Publications and source records attributed to S Paris.

At least 73 records · Page 4Linked to original sources
PubMed1996

Increased nitric oxide formation in recurrent thrombotic microangiopathies: a possible mediator of microvascular injury.

The term thrombotic microangiopathy (TMA) has been used extensively to encompass hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, two syndromes of hemolytic anemia, and thrombocytopenia associated with renal or brain involvement or both. There is evidence that endothelial damage is a crucial feature in the sequence of events that precedes the development of microvascular lesions. More recent studies would suggest that endothelial dysfunction could be a consequence of neutrophil activation. Activated neutrophils generate superoxide anions (O2-) that, combining with endothelial-derived nitric oxide (NO), form the highly cytotoxic hydroxyl radical. Seven patients with recurrent forms of TMA and seven healthy volunteers were studied. Plasma concentrations of the NO metabolites, nitrites/nitrates, were elevated in the acute phase of TMA, indicating an increased NO synthesis in vivo. In addition, elevated serum concentrations of tumor necrosis factor, a potent inducer of endothelial NO synthase, were found in acute TMA. Serum from patients with acute TMA induced NO synthesis in cultured endothelial cells more than normal serum. Enhanced stimulatory activity was no longer found in the recovery phase. Release of O2- by neutrophils ex vivo was higher than normal in patients with acute TMA, but decreased in the recovery phase. Exactly the same trend was observed for plasma malondialdehyde and conjugated dienes, indicating that excessive oxygen radical formation in acute TMA is associated with increased lipid peroxidation. Thus, in recurrent forms of TMA, NO formation was increased as compared with controls. This was associated with signs of lipid peroxidation, likely the consequence of the interaction of NO with neutrophil-derived oxygen products.

3,4-Methylenedioxyamphetamine↗
PubMed1996

Bilateral nephrectomy stopped disease progression in plasma-resistant hemolytic uremic syndrome with neurological signs and coma.

Four women were admitted over three years because of anemia and renal failure. They had evidence of hemolytic uremic syndrome (HUS) with severe vascular involvement and glomerular collapse. Despite intensive plasma exchange, all patients developed neurologic signs (with seizures and coma in 2) and papilledema. Three developed refractory hypertension and three required dialysis. All patients had abnormal von Willebrand factor (vWF) fragmentation as reflected by decreased high molecular weight and increased low molecular weight vWF multimers in the circulation. Assuming that the disease was sustained by shear stress-induced abnormal vWF fragmentation in damaged renal microvasculature, bilateral nephrectomy was done. Surgery was followed within two weeks by complete hematologic and clinical remission consistently associated with the restoring of vWF fragmentation pathway to normal. We speculate that in HUS resistant to plasma exchange or infusion, removing the kidneys eliminates a major site of vWF fragmentation, which would limit platelet activation and protect patients from the further spreading of microvascular lesions.

Adult↗
PubMed1995

A soluble protein negatively regulates phospholipase D activity. Partial purification and characterization.

Phosphatidylcholine-specific phospholipase D (PLD) is an important signalling phospholipase in mammalian cells. Recently, PLD activity has been shown to be positively regulated by the GTP-binding protein ARF (ADP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytosol. The inhibitory factor is characterized as a large protein or a complex of proteins with a molecular mass higher than 300 kDa. Using permeabilized and pre-permeabilized HL-60 cells depleted of their cytosol, we demonstrate that the inhibitor acts on GTP[S]-stimulated PLD activity. This effect is immediate, persistent and dose dependent for GTP[S]-stimulated PLD. Different possibilities for a mechanism of action of the inhibitory factor on the regulation of GTP binding of ARF were investigated. This inhibitory factor is not the guanine-dissociating inhibitor (GDI) for the small G-binding proteins Rho (Rho-GDI), reported to be a PLD inhibitor, since specific antibodies against this protein did not recognize a protein in the peak containing the inhibitory factor for PLD activity. Furthermore, the inhibitory factor does not prevent the binding of GTP[S] to ARF in the presence of HL-60 membranes. This excludes its possible role as an inhibitor of an ARF/guanine exchange factor. The inhibitory factor not only inhibits a pathway of PLD through GTP[S] activation in particular of the small GTP-binding protein, ARF, but it also inhibits PLD activated via either protein kinase C (PKC) or tyrosine kinase activation. The inhibitory factor also decreases PLC activity and this effect seems to be secondary to the inhibition of PLD activity. We discuss a mechanism of action of the inhibitor on PLD and the importance of this enzyme activity for membrane traffic.

Animals↗
PubMed1995

The small G-protein ARF1GDP binds to the Gt beta gamma subunit of transducin, but not to Gt alpha GDP-Gt beta gamma.

AlF4- activates heterotrimeric G-proteins G alpha subunits but not small GDP/GTP-binding proteins like ARF1. On retinal membranes containing holotransducin (Gt alpha GDP-Gt beta gamma) and incubated with ARFGDP, AlF4- induced Gt alpha GDP-AlF4 release and ARFGDP binding, probably to the remaining membrane-attached Gt beta gamma. On phospholipid vesicles reconstituted with Gt beta gamma, ARFGDP bound in proportion to Gt beta gamma, and was released upon subsequent Gt alpha GDP addition. Thus ARFGDP competes with Gt alpha GDP for binding to Gt beta gamma, probably through a conserved motif in the 'alpha 2 helix' of Gt alpha and ARF. This motif is found in the C-terminal helix of PH domains that bind to G beta gamma.

ADP-Ribosylation Factor 1↗
PubMed1995

Myristoylation of ADP-ribosylation factor 1 facilitates nucleotide exchange at physiological Mg2+ levels.

Recombinant N-myristoylated bovine ADP-ribosylation factor 1 (myr-rARF1) has been expressed in bacteria and purified to near homogeneity with a high (85%) myristoylation efficiency. Myr-rARF1 and nonmyristoylated rARF1 have been compared with respect to their kinetics of guanine nucleotide exchange and their interactions with phospholipids. Myristoylation is shown to allow the release of bound GDP at physiological (mM) concentrations of Mg2+. GDP dissociation is slow in the absence of phospholipids but is accelerated 2-fold in the presence of phospholipid vesicles. On the contrary, myristoylation decreases 10-fold the rate of dissociation of GTP or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) in the presence of phospholipids. As a result, myr-ARF1 can be spontaneously activated by GTP or GTP gamma S (t1/2 approximately 30 min at 37 degrees C) at 1 mM Mg2+, in the sole presence of phospholipid membranes without the need for a nucleotide exchange factor. In contrast to the nonacylated protein, the GDP-bound form of myr-ARF1 interacts with phospholipids, as demonstrated by its cosedimentation with phospholipid vesicles and its comigration with phospholipid/cholate micelles on gel filtration. The interaction is, however, weaker than for the GTP-bound form, suggesting that only the myristate in myr-ARF1GDP interacts with phospholipids, whereas both the myristate and the amino-terminal hydrophobic residues in myr-ARF1GTP bind to phospholipids.

ADP-Ribosylation Factor 1↗
PubMed1995

[Functional limitation of the forearm after shaft fracture in childhood. Possible role of the antebrachial interosseous membrane: MRI and ultrasound studies].

Based on follow-up examinations of 16 patients aged 5 to 15 years, the integrity of the interosseous membrane following forearm shaft fracture was evaluated. The examinations which included magnetic resonance imaging (MRI) and ultrasound scanning of both the fractured and the nonfractured forearms were conducted 4 to 81 months after fracture. Using a 1.5 T MR tomograph, we obtained 20 cross-sectional images arranged over the entire length of the forearm which was in neutral position, while sonographicly at 7.5 MHz we obtained cross-sectional views of the proximal, middle and distal forearm thirds with the forearm in pronation, neutral position and supination at a time. The MR image of the interosseous membrane is a thin hypointense line, occasionally showing low contrast irregularities on the fractured side such as thickenings, tears or curvature inconstancies which would suggest membrane alterations. However, these alterations appear to be less than expected. They are not clearly seen in the ultrasound views, where the interosseous membrane is found as an echorich line. According to our preliminary results, there seems to be a correlation between MR-detected interosseous membrane alterations and initial fracture dislocation as well as functional fracture outcome, particularly forearm rotation.

Adolescent↗
PubMed1995

PAF mediates neutrophil adhesion to thrombin or TNF-stimulated endothelial cells under shear stress.

Platelet-activating factor (PAF) is known to modulate polymorphonuclear leukocyte (PMN) adhesion to endothelial cells cultured under static conditions and activated by thrombin. In contrast, there are no data on the role of PAF in PMN adhesion to cells exposed to flow conditions and activated by stimuli other than thrombin. Here we used the PAF receptor antagonist L-659,989 to evaluate PMN adhesion to human umbilical vein endothelial cells (HUVEC) in basal conditions or upon challenge with thrombin or tumor necrosis factor-alpha (TNF-alpha). Experiments were performed under dynamic flow using a parallel-plate flow chamber and a computer-based image analysis system. Rolling and adhesion of PMNs to endothelial cells significantly increased upon stimulation with thrombin. Thrombin-stimulated HUVEC also synthesized higher amounts of PAF than untreated cells. Pretreatment of PMNs with L-659,989 significantly reduced their rolling and adhesion to thrombin-activated HUVEC. Stimulation of HUVEC with TNF-alpha significantly increased the number of rolling and adherent PMNs as compared with untreated cells. Adhesion of PMNs to and migration across TNF-alpha-stimulated HUVEC were reduced by L-659,989, whereas cell rolling was unchanged. We conclude that PAF mediates leukocyte interaction under flow conditions with HUVEC activated by inflammatory stimuli.

Cell Adhesion↗
PubMed1994

Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus.

Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N-terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active-site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre-proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild-type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.

Amino Acid Sequence↗
PubMed1994

rodletless mutants of Aspergillus fumigatus.

Conidia of Aspergillus fumigatus adhere in vitro to host proteins and cells via the outer cell wall layer. The rodA gene of A. fumigatus was cloned by homology with the rodA gene of Aspergillus nidulans, which is involved in the structure of the rodlets characteristic of the surface layer. The A. fumigatus RODA protein sequence has 85% similarity to that of A. nidulans RODA; the sequence codes for a hydrophobin, a low-molecular-weight protein moderately hydrophobic and rich in cysteines. The gene was disrupted with the hygromycin B resistance gene. By transformation of protoplasts with the disrupted gene, RodA- mutants were generated. These mutants are deficient in the ability to disperse their conidia; their conidia lack the rodlet layer and are hydrophilic. The adhesion of the rodletless conidia to collagen and bovine serum albumin was lower than that of the wild type; in contrast, there was no difference between RodA- and RodA+ conidia in adhesion to pneumocytes, fibrinogen, and laminin, suggesting that RODA is not the receptor for these cells and proteins. RodA- conidia were pathogenic for mice.

Adhesiveness↗
PubMed1994

[Tools, progress and questions in the molecular study of Aspergillus fumigatus and invasive aspergillosis].

Development of A. fumigatus in the host tissues is due to the intrinsic biological characteristics of this fungus and to the impairment of the cellular defence reactions of the host. However, even today the understanding of the factors governing the infectivity of A. fumigatus remains very limited. For example, the cellular mechanisms involved in the killing of A. fumigatus are still not elucidated. The cellular site(s) of infection and the role of the different lung epithelia in the establishment of the fungus are unknown. No specific fungal virulence factors have been identified until now. Molecular biology techniques are powerful tools to investigate the pathogenesis of invasive aspergillosis. Recent developments in the study of this mycosis are presented in this review.

Aspergillosis↗
PubMed1994

Splenic abscess.

The character and management of splenic abscess has changed in the past decade. The condition is more frequent, diagnosis is more easily established, and survival is more likely. Seven patients with splenic abscess from 1981-1992 were retrospectively reviewed. These patients had different etiologies for their splenic abscess, including hematogenous bacteria spread, contiguous spread, and previous history of trauma to the spleen or left upper quadrant. Most patients presented clinically with fever, left upper quadrant tenderness, and leukocytosis. All patients underwent CT scanning that was reliably diagnostic. All seven patients underwent splenectomy. Six of the seven patients were discharged from the hospital. We conclude that CT scan remains the gold standard for definitive diagnosis of splenic abscess, and splenectomy is very effective therapy.

Abscess↗
PubMed1993

Myristoylation is not required for GTP-dependent binding of ADP-ribosylation factor ARF1 to phospholipids.

Membrane binding of ADP-ribosylation factors (ARFs) is GTP-dependent and seems to require amino-terminal myristoylation. Recently it has been proposed that myristoylation is needed not for the activation of ARF by GTP but for its subsequent association to membranes. Here we show that unmyristoylated bovine ARF1, expressed in bacteria, can be efficiently loaded with GTP gamma S (guanosine 5'-O-(thio)triphosphate) at 1 microM free Mg2+, in the presence of phospholipids. Unmyristoylated ARFGTP gamma S cosediments with phospholipid vesicles and totally binds to phospholipid-cholate micelles, as seen by gel filtration chromatography. We therefore propose that, in vivo, myristoylation is required for the interaction of ARFGDP with its membrane-bound exchange factor rather than for the association of ARFGTP with lipid membranes. Phospholipid-bound ARFGTP gamma S can also stably interact with and activate the catalytic subunit of cholera toxin, suggesting that ARFGTP provides a membrane anchor for cholera toxin and thereby facilitates its access to membrane-bound substrates.

ADP-Ribosylation Factor 1↗
PubMed1993

A transformant of Aspergillus fumigatus deficient in the antigenic cytotoxin ASPFI.

The aspfI gene encoding a ribonucleotoxin, a putative virulence factor of Aspergillus fumigatus, was inactivated by gene disruption. Gene replacement through homologous recombination by the disrupted allele tagged by the hygromycin B resistance marker was performed by transformation of a pathogenic strain. One transformant with the disrupted aspfI gene failed to produce the ASPFI protein and was shown to be pathogenic for mice. We concluded that this ribotoxin is not a main factor in the colonization of the lung tissues by A. fumigatus.

Allergens↗
PubMed1993

Virulence of alkaline protease-deficient mutants of Aspergillus fumigatus.

The gene encoding the secreted alkaline protease, a suspected virulence factor of Aspergillus fumigatus, was inactivated by gene disruption. The disruption was performed by transformation of a pathogenic strain of the fungus with a linear DNA fragment carrying the gene from which the central part was replaced by the selectable Escherichia coli hygromycin B dominant resistance marker. Two transformants were shown to produce no alkaline protease. Restriction fragment analysis of the DNA of these two transformants was consistent for chromosomal integration of the disrupted gene by homologous recombination. Both isogenic alkaline protease-producing and non-producing A. fumigatus strains invaded lung tissues, causing comparable mortality in immunosuppressed mice. A significant residual proteolytic activity observed in alkaline protease non-producing strain cultures could play a role in the invasion of the tissues by the fungus.

Animals↗
PubMed1993

Identification of the 33-kDa alkaline protease of Aspergillus fumigatus in vitro and in vivo.

Aspergillus fumigatus produced a 33-kDa serine protease (ALP) in vitro and in vivo. In vitro, this alkaline protease was secreted when the fungus was cultivated in the absence of protein, if the pH of the medium remained close to neutrality. Western blotting and immunoelectronmicroscopy studies showed that ALP was localised in the wall of the fungus and was degraded after secretion in the culture medium under conditions of low pH. Although present in the lung during infection, ALP did not appear to be diagnostically useful and was different from the precipitating chymotrypsin antigen used in the diagnosis of aspergilloma.

Animals↗
PubMed1993

Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatus.

A metalloprotease (MEP) secreted by Aspergillus fumigatus was isolated from an alkaline protease-deficient mutant after the fungus was cultivated in the presence of collagen as the sole nitrogen and carbon source. The enzyme was purified 50-fold from the culture supernatant after adsorption to hydroxylapatite and carboxy-methyl-Sephadex and after gel filtration. The molecular mass was determined to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was estimated at pH 5.5 by isoelectric focusing. Reducing agents and divalent cations strongly inhibited enzyme activity, whereas nonionic detergents had no effect. A. fumigatus MEP was totally inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. MEP is not able to cleave elastin and is thermosensitive. Sera from patients suffering from aspergilloma reacted with MEP in Western blotting (immunoblotting) analyses, suggesting that MEP promotes an antigenic response in these patients.

Antibodies, Fungal↗
PubMed1993

Cell wall antigens in Aspergillus fumigatus.

The Aspergillus cell wall contains most of the antigens secreted by the fungus during its active in vitro or in vivo growth. These antigens, which bind to the IgE and IgG of allergic and aspergilloma patients or are secreted in the biological fluids of patients with invasive aspergillosis, are of primary importance in the diagnosis of aspergillosis. Located at the interface between host and pathogen cells, the fungal cell wall plays a major role during fungal invasion. It contains several surface receptors involved in adhesion of the fungus to host proteins and cells. Some of the wall antigens are also directly involved in the colonization of the host tissues by the fungus. Very few of these putative virulence factors have been purified until now. A 33-kDa alkaline protease of the subtilisin family can hydrolyze several extracellular matrix proteins such as collagen, fibrinogen, elastin. However, gene disruption experiments have shown that protease-deficient mutants are still able to infect mice. An 18-kDa antigen, which has been detected in the urine of patients with invasive aspergillosis, is present in vivo in the lung of mice infected with A. fumigatus. It has a ribonuclease activity that cleaves a single phosphodiester bond in a highly conserved region of the ribosomal RNA. Its role in the virulence of A. fumigatus has not been demonstrated until now. Biochemical and molecular characterization of the wall antigenic aggressins should be pursued.

Allergens↗