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Biomedical subjects

R Hellio

Publications and source records attributed to R Hellio.

At least 19 recordsLinked to original sources
PubMed1995

Targeting of Listeria monocytogenes ActA protein to the plasma membrane as a tool to dissect both actin-based cell morphogenesis and ActA function.

Actin assembly on the surface of Listeria monocytogenes in the cytoplasm of infected cells provides a model to study actin-based motility and changes in cell shape. We have shown previously that the ActA protein, exposed on the bacterial surface, is required for polarized nucleation of actin filaments. To investigate whether plasma membrane-associated ActA can control the organization of microfilaments and cell shape, variants of ActA, in which the bacterial membrane signal had been replaced by a plasma membrane anchor sequence, were produced in mammalian cells. While both cytoplasmic and membrane-bound forms of ActA increased the F-actin content, only membrane-associated ActA caused the formation of plasma membrane extensions. This finding suggests that ActA acts as an actin filament nucleator and shows that permanent association with the inner face of the plasma membrane is required for changes in cell shape. Based on the observation that the amino-terminal segment of ActA and the remaining portion which includes the proline-rich repeats cause distinct phenotypic modifications in transfected cells, we propose a model in which two functional domains of ActA cooperate in the nucleation and dynamic turnover of actin filaments. The present approach is a new model system to dissect the mechanism of action of ActA and to further investigate interactions of the plasma membrane and the actin cytoskeleton during dynamic changes of cell shape.

Actins↗
PubMed1995

Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor alpha, beta, and gamma chains.

Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and IL15 have common beta and gamma chains, while those for IL2, 4, 7, and 9 have a common gamma chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the alpha, beta, and gamma chains had different intracellular localizations after being endocytosed together. The alpha chain was always in transferrin-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the beta and gamma chains were excluded from transferrin-positive organelles and did not colocalize with alpha. Furthermore, beta could be found in rab7-positive vesicles. These differences suggest that the alpha chain recycles to the plasma membrane, while the beta and gamma chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The beta and gamma chains are very short-lived polypeptides since their half-life on the surface is only approximately 1 h, whereas alpha is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.

Antibodies, Monoclonal↗
PubMed1995

Intracellular Leishmania amazonensis amastigotes internalize and degrade MHC class II molecules of their host cells.

In their amastigote stage, Leishmania live in mammalian macrophages within parasitophorous vacuoles (PV), organelles of phagolysosomal origin that, in macrophages activated with IFN-gamma, contain major histocompatibility complex (MHC) class II molecules apparently devoid of invariant chains. We have now studied the fate of PV-associated class II molecules in mouse bone marrow-derived macrophages infected with L. amazonensis amastigotes using immunocytochemical and biochemical approaches. We have found that at least a part of these class II molecules was internalized by amastigotes and reached structures very often located in their posterior poles. This process was much more obvious if infected macrophages were incubated with protease inhibitors like antipain, chymostatin, Z-Phe-AlaCHN2 and Z-Phe-PheCHN2, or if amastigotes were pre-treated with the irreversible cysteine protease inhibitor Z-Phe-AlaCHN2 before infection, clearly indicating that amastigotes also degraded the internalized class II molecules. Study of infected macrophage cryosections by immuno-electron microscopy allowed the identification of the class II-positive structures in amastigotes as the lysosome-like organelles known as megasomes. Other PV membrane components like the prelysosomal/lysosomal glycoproteins Igp110, Igp120 and macrosialin could not be detected in megasomes of amastigotes even after treatment of macrophages with protease inhibitors, suggesting the involvement of some specific mechanism(s) for the internalization of class II molecules. Interestingly, after treatment of infected macrophages with various protease inhibitors (antipain, leupeptin, E-64, Z-Phe-AlaCHN2, Z-Phe-PheCHN2), PV membrane as well as megasomes of amastigotes become positive for invariant chains. A quantitative analysis of amastigote-associated class II molecules based on enzyme immunoassays showed that: (a) amastigotes extracted from macrophages treated with both IFN-gamma and antipain or Z-Phe-AlaCHN2 contained a much greater amount of class II than amastigotes extracted from macrophages treated with IFN-gamma alone; (b) class II molecules associated with the former were mainly intracellular and, at least some of them, were complexed with invariant chains or fragments of invariant chains; (c) amastigotes pre-incubated with Z-Phe-AlaCHN2 before infection accumulated a greater amount of intracellular class II than amastigotes pre-incubated without inhibitor, clearly indicating that the blockade of parasite cysteine proteases was sufficient to enhance the pool of these molecules within megasomes. On the whole, these data are consistent with the idea that class II molecules reaching PV are newly synthesized and still complexed with intact invariant chains or with partially degraded invariant chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Shigella flexneri enters human colonic Caco-2 epithelial cells through the basolateral pole.

The commonly accepted view that enteroinvasive bacteria enter cells of the intestinal epithelial lining through the apical surface can be challenged in the case of shigellosis. This study is based on in vitro experiments that showed that the invasion of human colonic Caco-2 cells by Shigella flexneri occurred through the basolateral pole of these cells. In these experiments, the few bacteria that interacted with the apical surface either bound to microvilli of the cell dome without causing detectable alteration or bound at the level of intercellular junctions at which they demonstrated a limited capacity for paracellular invasion, which permitted subsequent entry through the lateral domain of the cells. Treatment of Caco-2 cell monolayers with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), which disrupts intercellular junctions, greatly enhanced the rate of cell infection. These observations suggest a physiopathological paradox that may have important consequences for the understanding of the process of colonic invasion in vivo during shigellosis.

Bacterial Adhesion↗
PubMed1992

Unipolar reorganization of F-actin layer at bacterial division and bundling of actin filaments by plastin correlate with movement of Shigella flexneri within HeLa cells.

Shigella flexneri causes bacillary dysentery, an invasive disease of colonic epithelial cells in humans. The capacity of bacteria, once they have entered into a cell and escaped the phagocytic vacuole, to spread intracellularly and directly to adjacent cells without further extracellular passage is a key factor in invasion of the epithelial layer. Movement of intracellular bacteria is dependent upon the polymerization of actin; concentration of the formed filaments to one end of the bacterium is associated with initiation of movement. This movement may lead to the formation of a protrusion at the cell surface through which the bacterium passes to an adjacent cell. Development of these protrusions in infected HeLa cells is described, with emphasis on two critical observations. First, initiation of movement is coupled with bacterial division since elongation of the bacterial body is associated with relocalization of the previously uniformly distributed layer of actin to one pole of the bacterium. Second, the actin-bundling protein plastin appears to bundle the actin filaments just posterior to the bacterium, producing an ongoing contraction of the cylindrical actin tail that may be associated with forward movement of the bacterium within the protrusion.

Actins↗
PubMed1992

Movement along actin filaments of the perijunctional area and de novo polymerization of cellular actin are required for Shigella flexneri colonization of epithelial Caco-2 cell monolayers.

Shigella flexneri invades eucaryotic cells and grows in the cytoplasm. Lysis of the phagosomal membrane is a prerequisite for both intracellular multiplication and movement of the bacteria that gain direct access to the host cell actin. In HeLa cells, bacteria generate their own movement essentially by inducing actin polymerization. Polymerization of actin enables them to move rapidly and randomly in the cytoplasm and to spread from one cell to another through protrusions of the host cell membrane. This movement was designated the Ics phenotype. In contrast, in chicken embryo fibroblasts, bacteria move along actin filaments in a very organized manner, following the cytoskeletal architecture; this movement was designated the Olm phenotype. Bacterial movement is a major virulence factor in that it is necessary for efficient colonization of the intestinal epithelium of infected macaque monkeys. Further characterization of the cellular events that lead to colonization of the colonic intestinal epithelium was needed. In order to characterize the movement in vitro in a cell assay system more closely related to the intestinal epithelium, we used human colonic epithelial Caco-2 cells. The movement of bacteria as observed by using immunofluorescence and confocal microscopy appeared to result from the expression of both the Olm and Ics phenotypes. The former allowed colonization of cells along the actin filament ring of the perijunctional area. The latter promoted passage from one cell to adjacent cells. This in vitro pattern of movement and multiplication gives S. flexneri, once it has entered an epithelial cell, the unique capacity to spread through the entire epithelial layer without having further contact with the extracellular compartment.

Actin Cytoskeleton↗
PubMed1991

A new insight into the mycobacterial cell envelope architecture by the localization of antigens in ultrathin sections.

In an attempt to have a better insight into the mycobacterial cell envelope architecture, various subcellular fractions of Mycobacterium avium were prepared and characterized chemically and ultrastructurally. The various fractions corresponding to the mycobacterial "capsular material", outer layer, cell wall skeleton, cytoplasmic membrane, and cytosol as well as intact bacteria were then used to raise antisera in rabbits. The antisera so raised were then used to immunolabel the intact bacteria prior to embedding in epon. In parallel studies, bacteria were processed by a novel gelatin-uranyl acetate-low temperature Lowicryl HM20 embedding which preserved mycobacterial antigens, permitting to immunolabel antigens on ultrathin sections. Immunolabelling of epon-embedded intact bacteria showed that in the tripartite structure of the bacterial cell envelope, the middle electron-transparent layer acted as a barrier, not permitting the antibodies to penetrate into deeper structures. Immunolabelling of ultrathin sections showed that mycobacteria were surrounded by a "capsule" containing specific surface antigens with a glycocalyx-like topography, and that the intermediate electron transparent layer which separated the surface amphiphils from the inner arabinogalactan-peptidoglycan layer, was a virtual no man's land as it only seldom contained a single gold particle irrespective of the various antisera used. Furthermore, location of various layers in the cell envelope of M. avium using antisera raised against the subcellular fractions prepared was in agreement with chemical and ultrastructural data. A cell envelope model compatible with chemical, ultrastructural and immunolabelling data is proposed and its validity discussed.

Antigens, Bacterial↗
PubMed1991

Stress fiber-based movement of Shigella flexneri within cells.

icsA (virG), a gene located on pWR100, the virulence plasmid of Shigella flexneri serotype 5 (M90T), encodes a 120-kDa outer membrane protein. This protein promotes a random intracellular movement of the bacteria and leads to the infection of adjacent cells by the formation of protrusions. This movement, which involves the nucleation, polymerization, and subsequent polarization of actin, is referred to as the Ics phenotype (intra/intercellular spread). Here we present evidence that a second, distinct form of locomotion is also elaborated by S. flexneri in chicken embryo fibroblasts in which the Ics phenotype is not expressed. Using a combination of phase-contrast microcinematography and confocal microscopy, we have demonstrated that bacteria adopt parallel orientations by interacting with stress fibers. This interaction subsequently results in bacterial movement along the stress fibers themselves. This phenomenon occurs independently of the presence of a phagocytic vacuole which is lysed shortly after entry of the bacteria into the cell. It is expressed by M90T and SC560, its icsA mutant. This movement has been termed organelle-like movement (Olm phenotype) and is thought to account for the early accumulation of bacteria seen near the nucleus.

Actins↗
PubMed1991

Differential handling of bacterial antigens in macrophages infected with Mycobacterium leprae as studied by immunogold labeling of ultrathin sections.

Mycobacterium leprae were purified from the livers of experimentally infected armadillos, and the purity of the bacterial preparation was established by electron microscopy, immunoelectrophoresis of purified bacilli with rabbit serum raised against liver tissues from a noninfected armadillo, and gas chromatography. Such purified and intact bacilli were fixed and embedded by a gelatin-Lowicryl method for electron microscopy which preserved the mycobacterial antigens. Ultrathin sections were labeled with antisera raised in rabbits against the total antigens of the following species of mycobacteria: M. leprae, M. bovis BCG, M. avium, and a rapid-growing, nonpathogenic species, M. fallax. Bacteria were also labeled using serum raised against 2,3-diacyl-trehalose-2'-sulfate (sulfolipid-IV or SLIV) isolated and purified from M. tuberculosis. The immunolabeling was visualized under the electron microscope (EM) by using a secondary probe (goat-antirabbit IgG, H+L, coupled to 5 nm gold particles; GAR-5). EM results showed that M. leprae bacilli were highly labeled with all of the antisera used except SLIV, which was present only in discrete amounts. All of the antisera used labeled the bacterial "capsule," showing that this structure was not an artifact since it contained mycobacterial antigens. In parallel experiments, the murine J-774 macrophage cell line was infected with purified M. leprae, and fixed for EM at various time intervals for 1 week. Although the phagocytized bacteria did not multiply during the 1-week experiment, macrophages were unable to lyse them. Immunogold labeling of bacterial antigens in ultrathin sections of infected macrophages helped us to conclude: a) bacterial death and/or lysis is not a prerequisite for processing of antigens by infected macrophages; b) there was conclusive evidence for a differential antigen handling, i.e., some antigens were rapidly released (within 2 days, mostly capsular antigens) inside infected macrophages and transported to the macrophage surface, whereas others (the majority of them located in the cell-wall skeleton and in deeper bacterial structures) remained unreleased even after 4 to 7 days of infection; c) although relatively fewer epitopes reacting with anti-SLIV antibodies were found, they were rapidly released (within 2 days) inside macrophages, and exocytized to the macrophage surface. These novel findings are discussed in relation to leprosy and the current knowledge about the processing of bacterial antigens.

Animals↗
PubMed1990

Evidence that the capsule around mycobacteria grown in axenic media contains mycobacterial antigens: implications at the level of cell envelope architecture.

The intracellular growth of pathogenic mycobacteria has been linked to the presence of an electron transparent zone (ETZ or capsule), which surrounds the phagocytized bacteria and prevents the diffusion of lysosomal enzymes in infected macrophages. Recently, it was suggested that this capsule may be a bacterial structures, even being present in test tube-grown pathogenic mycobacteria (FEMS Microbiol. Lett. 1988, 56, 225-230). In the present paper, we show that under special fixation and embedding conditions, this capsule was clearly observed among 7 strains of mycobacteria grown in axenic media and also in M. leprae extracted and purified from experimentally infected armadillo or nude mice. In the case of bacteria treated likewise but subject to a prior dehydration step, this capsular structure disappeared suggesting its lipidic nature. Ultrathin sections of M. intracellular after immunolabelling showed for the first time that this capsule obtained mycobacterial antigens confirming its mycobacterial origin. It is suggested that the mycobacterial capsule may be formed of inert lipids, in which surface antigens are embedded.

Antigens, Bacterial↗
PubMed1990

Modified lysosomal compartment as carrier of slowly and non-degradable tracers in macrophages.

A previous immunocytochemical study of macrophages infected with Bacillus subtilis showed that a cell wall antigen could be detected for several days in a population of small vesicles randomly distributed within the cells and apparently distinct from perinuclear lysosomes. These observations suggested the possibility that these vesicles might constitute a "storage" compartment for non-degradable compounds. In the present report we compared in pulse-chase experiments the location and fate of a series of degradable and non-degradable pinocytic tracers within the macrophages. The tracers, detected by fluorescent microscopy, were bovine serum albumin (BSA), hen egg ovalbumin (OVA), horseradish peroxidase (HRP). Lucifer Yellow, fluorescent dextran, and levan. BSA and OVA remained located in perinuclear lysosomes during the chase period until their disappearance occurring within 3 h. In contrast, the other tracers, although initially located in perinuclear lysosomes, were found after a 3 to 5-h chase in small vesicles homogeneously distributed in the macrophage cytoplasm where they remained visible for 2 to 3 days. The use of markers for different cell organelles indicated that these dispersed vesicles exhibited several of the lysosomal features. They were acidic, they contained the 100 kDa and the 120 kDa lysosomal proteins as well as some acid proteases albeit these markers were in lesser concentrations than in the perinuclear lysosomal compartment. The addition of bacteria to the macrophages previously loaded with fluorescent dextran showed that all dispersed vesicles have the same fusion property as lysosomes and that slowly degraded or non-degradable tracers turn over through the perinuclear lysosomal compartment by using the endocytic pathway. Measurement of the release of some of the tracers into the culture medium suggested that the "dispersed vesicles" were probably not implicated in exocytosis of the tracers.

Animals↗
PubMed1988

Nutritionally variant streptococci develop ultrastructural abnormalities during experimental endocarditis.

Nutritionally variant streptococci (NVS) are fastidious micro-organisms responsible for most of the so-called negative blood culture endocarditis. In patients, the relative resistance of these bacteria to antibiotic treatment may be relevant to bacterial alterations in infected tissues. We used here a rabbit experimental model of endocarditis in order to examine, under scanning and transmission electron microscope, the NVS ultrastructure inside cardiac vegetations and to follow alterations at the different stages of the disease. In the early phase (day 7) of endocarditis, NVS were found dispersed inside vegetations and exhibited a typical streptococcal morphology similar to that observed during an in vitro balanced growth. In contrast, on day 11 and day 18, bacteria were found gathered as large and numerous clusters, in which they exhibited abnormal ultrasturctural features similar to those previously described during in vitro unbalanced growth. At this stage, ruthenium red staining revealed a large amount of exopolysaccharide surrounding the bacteria. None of these bacterial alterations were observed inside the vegetations of rabbits treated from day 7 to day 11 with penicillin or vancomycin. These abnormalities might be mainly related to nutrient limitation inside the clusters and could contribute to the pathogenicity of these micro-organisms.

Animals↗
PubMed1985

Role of particle electrostatic charge in adhesion and ingestion in Dictyostelium discoideum amoeboid cells.

The use of colloid iron hydroxide, Alcian Blue and enzymic treatments confirmed that the negative charges of Dictyostelium discoideum cell surface are mostly due to carboxylic groups. The role of electrostatic charges in adhesion and ingestion was determined by exposing cells to untreated latex beads, polylysine or polyglutamic-acid-coated beads, or carboxylated beads. This study showed that negatively charged beads presented a weaker adhesion strength than positively charged ones and that adhesion was strongest with untreated beads. This shows that hydrophobic forces are stronger than those induced by opposite electric charges between particle and cell surface. The ingestion rate was not directly related to adhesion because negatively charged beads presented the highest phagocytic rate, polylysine-coated beads had the lowest and neutral beads an intermediate one. The treatment of cells with polylysine or polyglutamic acid before neutral bead addition generally enhanced the ingestion rate. Electron microscopic observations made on polylysine-treated cells showed, however, that this stimulating effect was not due to the presence of the polymer on the cell surface but to a membrane clearance phenomenon occurring during polymer elimination. All these observations have led to the conclusion that the electric charges of the particle do not play a major role in D. discoideum phagocytic ability. Hydrophobic forces and probably other unknown parameters related to the cell surface properties are more important.

Cell Adhesion↗
PubMed1980

Electron-microscope study of Dictyostelium discoideum plasma membrane and its modifications during and after phagocytosis.

The study of plasma membrane and phagosome membrane of Dictyostelium discoideum was performed using 2 lectins: concanavalin A (Con A) and Wheat Germ Agglutinin (WGA), and 2 markers of anionic sites: colloidal iron hydroxide (CIH) and cationized ferritin (CF). These labellings were applied to fixed partially broken cells, which had ingested a large quantity of yeast. They showed that Con A and CF labelled both the outer and inner faces of the plasma membrane, whereas CIH and WGA were deposited on the outer face only. Phagosome membranes displayed the same location as the plasma membrane for Con A, CF and CIH even in very old phagosomes. This suggests that most receptors of these 3 markers were not degraded by hydrolases. In contrast, phagosome membranes of young and old phagosomes did not react with WGA. When labellings were made on yeast phagocytozing cells, the membrane of phagocytic cups were also devoid of WGA, while it was labelled with the 3 other markers. The absence of WGA labelling was not observed during ingestion of bacteria and latex beads, suggesting a specific relationship existed between yeast cells and WGA receptors.

Binding Sites↗
PubMed1980

Relationships between anionic sites and lectin receptors in the plasma membrane of Dictyostelium discoideum and their role in phagocytosis.

The disappearance of Wheat Germ Agglutinin (WGA) receptors from the membrane of yeast-engulfing-phagocytic cups in Dictyostelium suggested that these receptors could play a role in yeast adsorption or ingestion. This problem was approached by comparing the fate of WGA, Concanavalin A (Con A) and cationized ferritin (CF) and their effects on the phagocytosis of yeast, bacteria and latex beads. It can be concluded that CF capped in about 30 min and inhibited phagocytosis of any kind of particles for about 15 min. Con A capped in 20--60 min and inhibited phagocytosis of all particles for 1 h 30 min. The time at which phagocytosis started to occur corresponded approximately to the moment at which large areas of plasma membrane were totally devoid of marker. WGA did not cap but induced the formation of large and tight aggregates. The surface of the peripheral cells progressively released WGA in 1 h 30 min. Afterwards, the cells were able to ingest latex beads and bacteria but did not phagocytoze yeast. The latter started to be adsorbed onto the cells and to be ingested only 1 h later. Double labelling experiments showed that CF and Con A receptors were still absent in the plasma membrane, when phagocytosis of any kind of particles started to occur. WGA-labelled cells ingested latex beads and bacteria when their plasma membrane was still devoid of WGA receptors but were able to ingest yeast only after their regeneration. These observations strongly suggest that WGA receptors may correspond to specific receptors for yeast phagocytosis.

Binding Sites↗
PubMed1975

Physical studies of a plasmid mediating tetracycline resistance and hydrogen sulfide production in Escherichia coli.

Tetracycline resistance and hydrogen sulfide production have been previously found to be plasmid-mediated in a naturally-occurring strain of Escherichia coli; both functions are specified by a single conjugative plasmid called pIP231(Te-H2S); pIP231DNA was isolated as covalently closed molecules in dye-buoyant density gradients. The base ratio of the DNA was found to be 50% GC by density gradient analysis. Electron micrographs of plasmid molecules showed a contour length of 20 +/- 2 mum (40 +/- 4 X 10(6) daltons). Between one and two copies of pIP231 molecules per host chromosome were found in E. coli K12.

Base Sequence↗
PubMed1975

[Preliminary studies on chromatin subunits by freeze-etching].

We show that freeze-etching electron microscopy preserves and visualizes chromatin structure at level 25 A. Chromatin depleted of histone H 1 is observed to consist of subunits each having a central asymmetric core of DNA and protein 100 by 120 A. Around this core is wound a loop of DNA containing about 130 base pairs and perhaps some protein.

Animals↗