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J Bouvier

Publications and source records attributed to J Bouvier.

At least 19 recordsLinked to original sources
PubMed2002

Transient repressor effect of Fis on the growth phase-regulated osmE promoter of Escherichia coli K12.

Transcription of the gene osmE of Escherichia coli is osmotically inducible and regulated by the growth phase. Expression of osmE is directed by a single promoter, osmE (p), which is recognized by Esigma(70) and Esigma(s), two forms of RNA polymerase using, respectively, the sigma factors sigma(70) and sigma(s). Esigma(s) transcribes osmE (p) during entry into stationary phase. Esigma(70) is responsible for osmotic induction of osmE (p) during the exponential growth phase. In a search for proteins that can modulate osmE (p) expression in trans, we performed electrophoretic mobility shift experiments using a DNA fragment carrying osmE (p) and crude extracts from E. coli. One major retarded band was observed in these experiments. The Fis protein is responsible for this retarded band, and binds to several sites upstream and downstream of, and overlapping, the promoter region of osmE. In a fis mutant background, the kinetics of in vivo transcription of osmE (p) during growth demonstrated that Fis is not responsible for the repression of the promoter seen during early exponential phase. In contrast, expression of osmE (p) at elevated osmolarity during the mid-exponential growth phase is increased in the absence of Fis, demonstrating that Fis is able to act as a repressor in vivo at a particular stage of growth.

Bacterial Proteins↗
PubMed2000

Characterization of OpuA, a glycine-betaine uptake system of Lactococcus lactis.

A Lactococcus lactis glycine-betaine transport system was identified by functional complementation of an Escherichia coli proP proU mutant with a gene library from L. lactis sbsp. cremoris. The cloned locus forms an operon highly homologous to opuA, encoding a glycine-betaine uptake system of Bacillus subtilis. Disruption of opuA in L. lactis abolished protection by glycine-betaine against elevated osmolarity. OpuA belongs to the so-called "ABC transporters" family, which comprise an extracellularly localized substrate-binding protein. In B. subtilis OpuA system, this binding protein is a lipoprotein, attached to the external face of the cytoplasmic membrane by its lipidic moiety. In contrast, in the L. lactis opuA operon, and in other gram-positive homologues as well, a fusion between the gene encoding the integral membrane protein and the substrate-binding protein components gave rise to a hybrid protein presumably attaching the substrate-binding protein to the surface of the cell via its covalent link to the integral membrane component. Mapping of L. lactis opuA transcription start identified one mRNA, more abundant in cells grown at elevated osmolarity. Construction of an opuA-gusA fusion confirmed that opuA transcription is directed by a promoter osmotically inducible in L. lactis. When recombined upstream from a lac transcriptional fusion in the chromosome of E. coli, the opuA promoter appeared as very strong, and only poorly stimulated by elevated osmotic pressure, suggesting the existence of a specific machinery involved in the osmotic signal transduction in L. lactis.

ATP-Binding Cassette Transporters↗
PubMed1999

Pathology findings with acrylic implants.

We report the pathological findings in cases of acrylic implants obtained by direct intratumoral injection of polymethyl-methacrylate (PMMA) and N-butyl-cyano-acrylate (NBCA). Direct intratumoral injection of acrylic implants was performed for a variety of primary and secondary bone lesions. These types of treatments have been used at our institution in the last 4 years for 40 vertebroplasty (PMMA) procedures and for nine bone lesions of other locations (PMMA, NBCA). Postmortem histology became available for 1 case of PMMA and for 5 cases with NBCA intratumoral acrylic implants. The pathological findings associated with PMMA and NBCA were evaluated and compared. PMMA exhibited a macroscopic and microscopic rim of tumor necrosis, 6 months after implantation. NBCA exhibited compressive effects on the nearby tumor tissue, however, without signs of significant necrosis outside the acrylic tumor cast. Tumor captured inside the acrylic cast showed extensive to near complete necrosis. Acrylic implants may lead to necrosis when injected directly in tumors. The necrotizing effect may extend beyond the limits of an implant in the case of PMMA. Such an extended effect of PMMA, when compared with NBCA, may be due to the variable toxicity of acrylic implants, including the different degrees of the exothermic reaction during polymerization.

Adenocarcinoma↗
PubMed1998

Interplay between global regulators of Escherichia coli: effect of RpoS, Lrp and H-NS on transcription of the gene osmC.

The transcription of the osmC gene of Escherichia coli is regulated as a function of the phase of growth. It is induced during the decelerating phase, before entry into stationary phase. osmC expression is directed by two overlapping promoters, osmCp1 and osmCp2. osmCp2 is mainly transcribed by E-sigma(s), the RNA polymerase using the sigma(s) (RpoS) sigma factor, and is responsible for the growth phase regulation. Transcription from osmCp1 is independent of sigma(s). The leucine-responsive protein (Lrp) has been shown to bind the osmC promoter region in band shift experiments. In vivo analysis using osmC-lacZ transcriptional fusions demonstrated that Lrp affects the expression of both promoters. It represses the transcription of osmCp1 and activates the transcription of osmCp2 by E-sigma(s). An absence of Lrp results in an increase in the amount of RpoS during exponential growth in minimal medium. The nucleoid-associated protein H-NS also represses osmC transcription from both promoters. However, this happens through different mechanisms. The effect on osmCp2 is probably mediated by the increase in sigma(s) concentration in the cytoplasm of hns- mutants, while the effect on osmCp1 is independent of sigma(s). No binding of H-NS to the promoter region DNA could be detected, indicating that the effect on osmCp1 could also be indirect.

Artificial Gene Fusion↗
PubMed1997

Incorporation and Antibody Recognition of a Lipid-Anchored Membrane Protein in Supported Lipid Layers

The formation of supported lipid layers incorporating promastigote surface protease (PSP), a glycosylphosphatidylinositol-anchored protein, is investigated using surface plasmon resonance. Both hydrophilic and hydrophobic substrates are used for the formation of lipid layers, and results are consistent with the formation of lipid bilayers and monolayers, respectively. Specific antibody binding to layers containing PSP is observed, whereas nonspecific binding of the antibody to the surface is effectively suppressed by the phosphatidylcholine lipid layer. Phosphatidylinositol-specific phospholipase C is used to remove the lipid moieties from the membrane-incorporated PSP, releasing it into solution in a hydrophilic form and demonstrating that a large fraction of the protein is anchored in the lipid layer via the lipid moieties. Copyright 1997 Academic Press. Copyright 1997Academic Press

Journal Article↗
PubMed1997

Leishmania major: comparison of the cathepsin L- and B-like cysteine protease genes with those of other trypanosomatids.

Cysteine proteases play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As a first step toward applying this strategy to design inhibitors as antiparasitic agents for leishmaniasis, we have isolated and sequenced the full-length clones of two cysteine protease genes from Leishmania major. One of the genes is structurally similar to the cathepsin L-like family and the other is similar to the cathepsin B-like family of cysteine proteases. The L. major cathepsin L-like sequence has a proregion that shares high sequence similarity with other cathepsin L sequences but not cathepsin B sequences and has a proline/threonine-rich C-terminal extension. The cathepsin L-like gene occurs in multiple copies, whereas there may be only one copy of the cathepsin B-like gene. Northern blot analyses show that both genes are expressed in the promastigote and amastigote stages, and pulse field gel electrophoresis revealed that the cathepsin L- and B-like genes are each found on two nonhomologous chromosomes. The L. major L-like amino acid sequence is 75% identical to the L. mexicana sequence, 74% identical to the L. pifanoi sequence, 47% identical with the Trypanosoma cruzi sequence, 47% identical with the T. congolense sequence, and 45% identical with the T. brucei sequence. L. major is one of two trypanosomatid species for which a cathepsin B-like gene has been identified and sequenced; its amino acid sequence is 82% identical to the one from L. mexicana. Tree inference based on distance and parsimony methods of kinetoplastid cathepsin L proteins yielded independent support for phylogenetic hypotheses inferred from analyses of ribosomal RNA genes. Because the cathepsin L locus has a high level of phylogenetic signal with respect to trypanosomatid taxa, this locus has great potential utility for investigating the evolutionary history of trypanosomatids and related organisms.

Amino Acid Sequence↗
PubMed1996

[Mucinous ductal ectasia. Apropos of 2 cases].

Mucinous ectasia of the pancreas ducts is a recently described cystic lesion which usually occurs in acute pancreatitis. Endoscopic retrograde pancreatography provides diagnosis. Mucinous ductal ectasia is considered to be a precancerous lesion. Surgical exeresis is recommended. Our two cases emphasize the difficulties encountered for differential diagnosis with other cystic lesions of the pancreas.

Aged↗
PubMed1994

Influence of dye and protein location on photosensitization of the plasma membrane.

Two membrane-photosensitizing dyes were used to investigate whether selected sites in the plasma membrane vary in their sensitivity to damage by singlet oxygen (1O2*) and, if so, what factors are responsible for the variation. The relative ability of Rose bengal (RB) and merocyanine 540 (MC540), both of which localize in the plasma membrane and produce 1O2*, to photosensitize five plasma membrane functions in P388D1 cells was evaluated. The five membrane functions assessed were: plasma membrane potential, proline transport, facilitated glucose diffusion, 5'-nucleotidase activity, and dye exclusion. Photosensitization efficiency by RB varied by a factor of 188 for these membrane functions, whereas for MC540 a range of only 24 was found. RB was a more efficient photosensitizer than MC540 but the relative efficiencies varied with the membrane function. The wide range of P50 values for RB suggests that it binds selectively to membrane sites where it causes damage with high efficiency; possibly a non-1O2* mechanism is involved. In contrast, MC540 photosensitized the three membrane functions involving integral membrane proteins about equally suggesting that differences are due to small variations in the distribution of MC540 in the plasma membrane and/or variations in the inherent reactivity of the membrane targets with 1O2*. The results indicate that the lability of membrane sites to photosensitization depends both on their inherent reactivity with 1O2* and the relative location of specific protein and dye molecules.

5'-Nucleotidase↗
PubMed1994

Granzyme A released upon stimulation of cytotoxic T lymphocytes activates the thrombin receptor on neuronal cells and astrocytes.

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg-Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As brain-infiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.

Amino Acid Sequence↗
PubMed1994

Naegleria fowleri: characterization of a secreted histolytic cysteine protease.

Naegleria fowleri is the etiologic agent of primary amebic meningoencephalitis, a rare but rapidly fatal disease of humans. It invades the central nervous system via nasal mucosa and cribriform plate. Once in brain tissue, the organism induces an acute hemorrhagic, necrotizing meningoencephalitis. We hypothesize that a protease released by the parasite contributes to tissue destruction and facilitates host invasion. Analysis of conditioned media of N. fowleri cultures revealed a major 30-kDa protease with substrate and inhibitor specificity consistent with cysteine proteases. Amino-terminal amino acid sequence of the purified enzyme showed it to be a thiol protease with homology to cathepsin L. It catalyzed the in vitro degradation of extracellular matrix and had a cytopathic effect on mammalian cells. Both ameba-induced matrix degradation and the cytopathic effect are inhibited by Z-Phe-Ala-fluoromethyl ketone, an irreversible cysteine protease inhibitor. Our results indicate that N. fowleri secretes a cysteine protease with the capacity to destroy host tissue. Naegleria gruberi, a nonpathogenic species, expresses a similar protease but, unlike its pathogenic relative, is not thermotolerant to temperatures above 30 degrees C.

Amino Acid Sequence↗
PubMed1994

Dual core and shell temperature regulation during sea acclimatization in Gentoo penguins (Pygoscelis papua).

Penguins are able to maintain a high and constant body temperature despite a thermally constraining environment. Evidence for progressive adaptation to cold and marine life was sought by comparing body and peripheral skin temperatures, metabolic rate, and thermal insulation in juvenile and adult Gentoo penguins exposed to various ambient temperatures in air (from -30 to +30 degrees C) and water (3-35 degrees C). Juvenile penguins in air showed metabolic and insulative capacities comparable with those displayed by adults. Both had a lower critical temperature (LCT) close to 0 degree C. In both adults and juveniles, the intercept of the metabolic curve with the abscissa at zero metabolic rate was far below body temperature. This was accompanied by a decrease in thermal insulation below LCT, allowing the preservation of a threshold temperature in the shell. However, this shell temperature maintenance was progressively abandoned in immersed penguins as adaptation to marine life developed, probably because of its prohibitive energy cost in water. Thus adaptation to cold air and to cold water does not rely on the same kind of reactions. Both of these strategies fail to follow the classical sequence linking metabolic and insulative reactions in the cold.

Acclimatization↗
PubMed1993

Targeted activation of CD8 cells and infection of beta 2-microglobulin-deficient mice fail to confirm a primary protective role for CD8 cells in experimental leishmaniasis.

CD8+ T cells play an important role in the immunologic control of intracellular pathogens, particularly viruses. Leishmania are obligate intracellular parasites of macrophages in the mammalian host, and previous studies using deletion of CD8+ cells by administration of mAb to infected animals have suggested a protective role for these cells. Two complementary approaches were used to define more carefully the role of CD8+ cells in leishmaniasis. In BALB/c mice susceptible to Leishmania major (L. major) infection, targeted activation of CD8+ T cells was attempted by immunization with nonapeptides derived from the conserved major outer surface protein of the organism, gp63, that contained the consensus binding motif for MHC class I H-2Kd molecules. Two of the nonapeptides induced CTL activity in subsequently infected BALB/c mice that could be elicited against P815 cells pulsed either with peptide or lysates of L. major. Purified CD8+ T cells from immunized mice had elevated levels of IFN-gamma mRNA transcripts as compared to unimmunized mice. Despite evidence for activation of CD8+ cells, none of the mice immunized with nine different peptides alone or in combination were protected from progressive disease. In a second series of experiments, beta 2-microglobulin deficient mice that lack CD8+ cells were infected with L. major and the course of infection monitored. These mice cured disease as rapidly as beta 2-m +/- and +/+ littermates, and cure was associated with comparable levels of IFN-gamma mRNA in the draining lymph node population. Neither of these approaches was able to confirm a substantive role for CD8+ T cells in the primary protective response to L. major.

Amino Acid Sequence↗
PubMed1993

Brugia pahangi: identification and characterization of an aminopeptidase associated with larval molting.

The third-stage larvae (L3) of the filarid parasite Brugia pahangi were surveyed for protease activity beginning with release from the mosquito vector through molting to the fourth-stage larvae (L4). A metalloaminopeptidase with a substrate preference for phenylalanine was released during the molting process. A screen of aminopeptidase inhibitors identified H-boroPhenylalanine-(pinacol) as highly effective, with a Ki of 1.3 x 10(-11) M. This peptidase inhibitor also arrested molting of L3 larvae in vitro at a concentration of 100 microM.

Amino Acid Sequence↗
PubMed1993

A fluorescent peptide substrate for the surface metalloprotease of Leishmania.

A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry 29, 10113-10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 x 10(6) M-1sec-1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.

Amino Acid Sequence↗
PubMed1993

The proteases and pathogenicity of parasitic protozoa.

Protozoan parasites are among the most prevalent pathogens worldwide. Diseases like malaria, leishmaniasis, amebiasis, and trypanosomiasis affect hundreds of millions of people. Recent advances in our understanding of the biochemistry and molecular biology of these organisms has focused attention on specific parasite molecules that are key to the parasite life cycle or the pathogenesis of the diseases they produce. One group of enzymes that plays myriad roles in these processes are the parasite-derived proteases. Different types of proteases are frequently expressed at different stages of the parasite life cycle to support parasite replication and metamorphosis. Intracellular parasites such as those that produce malaria and Chagas' disease express high levels of protease activity to efficiently degrade host proteins like hemoglobin. In other instances, such as infection with Entamoeba histolytica, the causative agent of amebiasis, proteases released by the parasite can damage host cells and tissues, contributing to host tissue damage and parasite invasion. Detailed studies of these enzymes have led to model systems for the study of parasite gene regulation, parasite metabolism, and the host-parasite interplay. In some instances, proteases appear to be promising targets for the development of new antiparasitic chemotherapy.

Amino Acid Sequence↗
PubMed1993

Cloning of a virulence factor of Entamoeba histolytica. Pathogenic strains possess a unique cysteine proteinase gene.

Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined.

Amino Acid Sequence↗