Biomedical subjects

A Ryter

Publications and source records attributed to A Ryter.

At least 37 records · Page 2Linked to original sources

PubMed1988

Endocytic membrane traffic with respect to phagosomes in macrophages infected with non-pathogenic bacteria: phagosomal membrane acquires the same composition as lysosomal membrane.

A morphometric analysis was made to study membrane traffic in bone marrow-derived macrophages, containing phagosomes with partially degraded Bacillus subtilis. Cell surface glycoproteins, labeled with radioactive galactose by terminal glycosylation, provided a covalent autoradiographic membrane marker. Membrane compartments were characterized in terms of cytochemical staining for horseradish peroxidase taken up by receptor-mediated endocytosis. The area, composition, and exchange rates of endocytic membrane compartments were measured as in a previous analysis for non-infected macrophages, devoid of phagosomes. In direct comparison with this earlier study, the present data allowed an assessment of the involvement of phagosomes in the interactions between endocytic membrane compartments. The presence of phagosomes led to a 30% reduction of lysosomal membrane area. The rate at which cell surface-derived label flowed into the lysosomal membrane pool was reduced by the same fractional amount. This suggested a linear relationship between flow rate and membrane area. The initial flow rate of label into phagosomes was higher than expected, based on their membrane area being only about 60% that of lysosomes. This rate could only be measured during the early phase of the experiments when phagosomes were younger, therefore displaying a fast exchange rate, reminiscent of the endosome compartment. However, steady-state conditions, at late times, strongly suggested that phagosomes with degraded contents finally acquire membrane of lysosomal origin. First, the composition of phagosome membrane became the same as that of lysosomes, remaining unchanged as compared to non-infected cells. Second, the membrane area of phagosomes amounted to the loss of lysosomal membrane area in infected cells.

Animals↗

PubMed1988

[A new sensitivity test for substances used as antiseptics or disinfectants. I. Biological studies].

In this first article, the authors present a fast method of determining the bactericide and bacteriostatic power of the germs, based on the disparition of the succino-dehydrogenase activity of a bacterium, objectified by a neotetrazolium chloride reaction. This reaction allows a colorimetric qualitative and quantitative determination of the activity of a preparation and the duration of its contact with the bacteria. This method allows a study in electronic microscopy which will be published in a second article.

Anti-Infective Agents, Local↗

PubMed1987

Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase.

This article describes the reconstitution in Escherichia coli of a heterologous protein secretion system comprising a gene for an extracellular protein together with its cognate secretion genes. The protein concerned, pullulanase, is a secreted lipoprotein of the Gram-negative bacterium Klebsiella pneumoniae. It is initially localized to the cell surface before being specifically released into the medium. E. coli carrying the cloned pullulanase structural gene (pulA) produces pullulanase but does not expose or secrete it. Secretion genes were cloned together with pulA in an 18.8 kbp fragment of K. pneumoniae chromosomal DNA. E. coli carrying this fragment exhibited maltose-inducible production, exposition and specific secretion of pullulanase. Transposon mutagenesis showed that the secretion genes are located on both sides of pulA. Secretion genes located 5' to pulA were transcribed in the opposite orientation to pulA under the control of the previously identified, malT-regulated malX promoter. Thus these secretion genes are part of the maltose regulon and are therefore co-expressed with pulA. Transposon mutagenesis suggested that secretion genes located 3' of pulA are not co-transcribed with pulA, raising the possibility that some secretion functions are not maltose regulated.

Base Sequence↗

PubMed1987

Leishmania mexicana: a cytochemical and quantitative study of lysosomal enzymes in infected rat bone marrow-derived macrophages.

The cellular localization and activity of the lysosomal enzymes acid phosphatase, trimetaphosphatase, and arylsulfatase were studied in rat bone marrow-derived macrophages infected with Leishmania mexicana amazonensis amastigotes. The specific activity of acid phosphatase normalized for protein content was similar in normal macrophages and in isolated amastigotes, whereas the latter were markedly deficient in trimetaphosphatase and arylsulfatase activities. It is thus likely that trimetaphosphatase and arylsulfatase activities detected in infected macrophages were of host cell origin. The activities of the three enzymes, assayed biochemically, varied independently in the infected macrophages. While arylsulfatase activity was unchanged after infection, the activity of acid phosphatase increased by 19, 40, and 94% at 6, 24, and 48 hr, respectively. Trimetaphosphatase activity rose only slightly during the first 24 hr after infection but increased by 74% at 48 hr. The rise in acid phosphatase activity could be accounted for only partially by multiplication of the amastigotes. Thus, as for trimetaphosphatase, these results suggest enhanced macrophage synthesis of acid phosphatase and/or reduced enzyme degradation by the infected macrophages. The reduction in host cell lysosomes previously described (Ryter et al. 1983; Barbieri et al. 1985) was confirmed but appearance of lysosomal enzyme activity in the parasitophorous vacuole is documented in the present report. Thus, Leishmania do not seem to reduce the amount and the activity of host lysosomal enzymes.

Acid Anhydride Hydrolases↗

PubMed1987

Plasmid-mediated early killing of eucaryotic cells by Shigella flexneri as studied by infection of J774 macrophages.

In Shigella flexneri a 220-kilobase plasmid encodes the ability to invade nonprofessional phagocytes by a mechanism similar to phagocytosis. In this report, the continuous macrophage cell line J774 was used to study the intracellular fate of both invasive and noninvasive strains. pWR100, the virulence plasmid of S. flexneri serotype 5, mediated very efficient and rapid killing of J774 macrophages, as measured by cellular detachment and uptake of trypan blue. For this to occur, the bacteria had to be within the cells, since the macrophages were protected by cytochalasin D. A battery of strains differing in their levels of Shiga toxin production showed that inhibition of protein synthesis by Shiga toxin, as measured by [35S]methionine incorporation into infected macrophages, was not required for early killing of cells. Damage to J774 macrophages rather correlated with the ability of invasive bacteria to rapidly and efficiently lyse the membrane of the phagocytic vacuole. The role of the release of bacteria within the cytosol for subsequent expression of cytotoxic activity is discussed, and mitochondria are proposed as a potential target for this activity.

Animals↗

PubMed1987

Contribution of immunogold labelling to study of the outer membrane of gram-negative bacteria.

The immunogold labelling technique is beginning to make a significant contribution to the study of several biological systems. In the case of Gram-negative bacteria, immunogold labelling performed either on whole cells or on thin sections has recently begun to facilitate the study of outer membrane biogenesis, the topology of membrane proteins, and protein export.

Antigens, Bacterial↗

PubMed1987

Exchange kinetics and composition of endocytic membranes in terms of plasma membrane constituents: a morphometric study in macrophages.

Intracellular membrane traffic, during endocytosis in mouse bone marrow-derived macrophages, was studied quantitatively by morphometric and kinetic analysis. Three functionally different markers were used: Horseradish peroxidase (HRP) served as a fluid-phase (FP) marker (1000 micrograms HRP/ml in the presence of mannan) or as a receptor-mediated (RM) membrane marker (25 micrograms HRP/ml) and, third, plasma membrane (PM) glycoconjugates, enzymatically labeled with [3H]galactose at the cell surface, served as a covalent membrane marker. The cell surface was labeled with [3H]galactose, followed by either FP or by RM uptake of HRP. The kinetics of the intracellular appearance of the markers were measured as the membrane area stained by HRP-reaction product and as the number of autoradiographic grains associated with these membranes. The following compartments were distinguished: PM, coated vesicles (VI), pinosomes or endosomes (VII), secondary lysosomes (VIII), and HRP-negative vesicles (EV). Tubular structures of VII became labeled with HRP only during RM uptake. The markers flowed first into VI and VII, and after 5 min into VIII. EV became labeled with the covalent membrane marker starting from 5 min. The ratio of autoradiographic grain number to HRP-stained membrane area remained constant with time although substantially different for the various compartments, viz. 100% (VI), 50% (VII and EV) and 30% (VIII) as compared to the PM (100%). This indicated that endosomes were only partially derived from internalized PM and that secondary lysosomes contained a substantial pool of PM constituents. The observed kinetics suggested that once every 30 to 40 min the entire PM was internalized, the bulk of which was recycled after 4 min from a prelysosomal compartment(s) leaving only 12 to 20% for recycling via membranes of secondary lysosomes after a residence time of 24 to 33 min.

Acid Phosphatase↗

PubMed1986

Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface.

The LamB protein is a trimeric integral outer membrane protein from Escherichia coli K12 which functions as a pore for maltodextrins and a receptor for bacteriophages. When inserted into two selected sites of LamB, a foreign antigen, the C3 epitope from poliovirus, was exposed at the cell surface with its normal antigenic properties. Since these genetic insertions did not affect in any essential way the routing, activity and folding of the LamB protein, we conclude that the two corresponding LamB sites are at the cell surface as predicted by our recent model. We discuss the implications of our results for the study of protein topology with a single epitope and the direct cloning and cell surface expression of epitopes of interest as well as the development of live vaccines or diagnostic tests.

Amino Acid Sequence↗

PubMed1986

Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis.

Virulent strains of Shigella flexneri invade HeLa cells with high efficiency. This crucial step in the pathogenic process is encoded by a 140-megadalton plasmid which induces phagocytosis of the bacteria by host cells. In this report we used pWR100, the virulence plasmid of S. flexneri serotype 5, and pHS4108, a 32-megadalton subclone of pWR100, to demonstrate that the plasmid is also responsible for rapid intracellular growth of the bacteria. The ability to replicate intracellularly was not correlated with induction of Shiga toxin. However, plasmid-mediated intracellular multiplication was strongly correlated with the ability of the bacteria to rapidly and efficiently lyse the phagocytic vacuole and replicate freely in the cytoplasm. Temperature-regulated plasmid-mediated contact hemolytic activity strongly correlated with both phagosomal membrane lysis and efficient intracellular multiplication. We propose this virulence plasmid-associated hemolysin to be an important factor in the invasion and proliferation of Shigella spp. in mammalian cells.

HeLa Cells↗

PubMed1986

A deletion that includes the segment coding for the signal peptidase cleavage site delays release of Saccharomyces cerevisiae acid phosphatase from the endoplasmic reticulum.

We studied ultrastructural localization of acid phosphatase in derepressed Saccharomyces cerevisiae cells transformed with a multicopy plasmid carrying either the wild-type PHO5 gene or a PHO5 gene deleted in the region overlapping the signal peptidase cleavage site. Wild-type enzyme was located in the cell wall, as was 50% of the modified protein, which carried high-mannose-sugar chains. The remaining 50% of the protein was active and core glycosylated, and it accumulated in the endoplasmic reticulum cisternae. The signal peptide remained uncleaved in both forms. Cells expressing the modified protein exhibited an exaggerated endoplasmic reticulum with dilated lumen.

Acid Phosphatase↗

PubMed1985

Relationship between ultrastructure and specific functions of macrophages.

The main function of the macrophages, which is to ingest and degrade any foreign molecules or particles penetrating the organism, appears in the development of the different structures implicated in endocytic activity. The macrophage's high endocytic property first appears in its irregular shape and the large number of extensions of the cell membrane, allowing the rapid capture of extra-cellular material. Adhesion between macrophage cell surface and molecules or particles is greatly enhanced by the presence of varied kinds of receptors: lectin-like receptors which bind specific sugars or highly specific receptors such as Fc and C3b receptors, which increase phagocytosis of opsonized microbes. The microbicidal properties reside in part in the production of superoxide anions which result from the activity of a NAD(P)H oxidase. This enzyme is located in the plasma membrane. Its activity could be demonstrated with a cytochemical method, on the cell surface and along the phagosome membrane. It is, however, very weak in resident macrophages and increases after stimulation or activation. The second kind of bactericidal property corresponds to cationic proteins located in lysosomes. After fusion between lysosomes and phagosomes, they contribute to microbe killing by permeabilizing microbe envelopes. Lysosomes, which contain diverse acid hydrolases and are responsible for the degradation of ingested material, play a crucial role in macrophage endocytic activity. Their number increases in parallel with endocytic activity during macrophage differentiation and is particularly high after ingestion of degradable material. Contrary to polymorphonuclear leukocytes, macrophage is very poor in granules containing peroxidase. The latter, which are rather abundant in monocytes, disappear during macrophage maturation. They do not seem thus to be implicated in macrophage microbicidal activity. Endocytosis is accompanied by rapid and intense exchanges between the different membrane compartments of the cell (plasma membrane, pinosomes or phagosomes, endosomes, lysosomes, Golgi apparatus, etc.). These exchanges seem to occur by transitory fusions between vesicles coming from different compartments, rapidly followed by their recycling to their original compartment. This system of membrane shuttle has been clearly observed after formation of phagosomes or pinosomes in which the internalized plasma membrane is recycled back to the cell surface within a few minutes after their formation. This membrane traffic is especially intense in macrophages, the endocytic activity of which is very high, but it also exists in all cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Shape and fine structure of nucleoids observed on sections of ultrarapidly frozen and cryosubstituted bacteria.

Very rapidly frozen cells of Escherichia coli and Bacillus subtilis were substituted at low temperature into acetone with 1% OsO4 and embedded in Epon. They showed ribosome-free spaces filled with globular and fibrillar material of up to 15 nm. The sizes of structures seen do not exclude DNA superstructures such as supercoils, aggregates, and nucleosomes. With the Feulgen analog osmium-ammines stain, DNA was localized within the ribosome-free space. The bulk of DNA, the nucleoid, is therefore a major part of, or identical to, the main ribosome-free space. The ribosome-free space would correspond directly to the light microscopy phase-contrast image of nucleoids in living bacteria. The shape of the ribosome-free space does not reflect intracellular salt concentrations, nor do the Feulgen-positive areas. The previously observed dependency on the salt concentration of the growth medium seems to be due to permeabilization induced by the chemical fixative at room temperature. The ribosome-free space is more cleft in appearance than the nucleoid obtained by fixation with OsO4 but more confined than its very dispersed form found after aldehyde fixation.

Bacillus subtilis↗

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↗

PubMed1984

Immunolabelling of bacteriophage lambda receptor protein (LamB) on thin sections of E. coli embedded in Lowicryl.

LamB is one of the major cellular proteins when E. coli is grown in the presence of maltose and is localized in the outer membrane. Previous immunolabellings obtained with monoclonal antibodies showed that this protein is a transmembrane protein and led to the detection of 4 epitopes exposed on the cell surface and 2 located on the inner surface of the outer membrane (Scheckman et al., 1983). In the present study, we have used this biological model in order to see whether these two classes of epitopes could be distinguished by immunocytochemical labelling performed on thin sections of E. coli embedded in Lowicryl K4M (Carleman et al., 1982). The optimal conditions of fixation and embedding were first established for labelling with poly- or monoclonal antibodies detected by Protein A-gold complexes. The analysis of gold particle distribution on each side of the outer membrane after labelling with a polyclonal serum or after its adsorption on intact bacteria allowed us to conclude that the resolution of immunolabelling on thin sections was about 20 nm. The use monoclonal antibodies met with difficulties due mostly to the nonspecific labelling of the cytoplasm. Although this nonospecific labelling was decreased by fixing bacteria with paraformaldehyde alone, only one antibody gave a correct specific labelling after high dilution (1/3000). The gold particle distribution obtained with this antibody confirmed the location on the cell surface of this epitope.

Antibodies, Bacterial↗

PubMed1984

[Not Available].

Explore the source record for details and available documents.

Contraception↗

PubMed1983

Leishmania mexicana: acid phosphatase ultrastructural cytochemistry of infected mouse macrophage cultures treated with phenazine methosulfate.

Bone marrow-derived mouse macrophage cultures infected with Leishmania mexicana amazonensis amastigotes were given a 2-hr pulse with 10 microM phenazine methosulfate (PMS), a cationic electron carrier which destroys the intracellular parasites. Cultures were fixed at different times after the PMS pulse and processed for the detection of acid phosphatase (AcP) activity at the electron microscopic level. Only a small proportion of nontreated, infected macrophages stained for AcP. In contrast, 2 to 6 hr after exposure to PMS, many infected cells displayed AcP-positive lysosomes and parasitophorous vacuoles. This increased AcP reactivity paralleled the reduction in the percentage of morphologically intact parasites. In addition, qualitative observations indicated that while nontreated infected cells contained only few recognizable lysosomes, the lysosomal complement noticeably increased a few hours after exposure to PMS. Most intact intracellular amastigotes were not stained, but damaged parasites were often positive for AcP. Twenty hours after the PMS pulse, the percentage of AcP-positive macrophages dropped to the levels initially present in noninfected cultures and all of the parasites were destroyed. Exposure of noninfected macrophages to PMS did not affect their AcP reactivity.

Acid Phosphatase↗

PubMed1983

Cytochemical study of the nucleolus of the slime mold Dictyostelium discoideum.

The nucleus of the slime mold Dictyostelium discoideum is characterized by the presence of several large dense masses which are all in tight contact with the nuclear membrane. These dense masses, considered as nucleoli, present a rather homogeneous texture, in which dense chromatin, fibrillar, and granular material are not easily detected. The autoradiographic study of [3H]uridine pulse-labeled cells showed that the majority of the silver grains were located inside these masses. The use of EDTA regressive-staining, acetylation and enzymatic digestion indicated that they are mostly composed of RNP and are totally devoid of dense chromatin as the rest of the nucleus is. After treatment with actinomycin D, fibrillar and granular material segregated but no chromatin could be found. All these observations confirmed that the dense masses correspond to nucleoli despite their peculiar ultrastructure. It can also be concluded that this type of nucleoli cannot be considered as a taxonomic character of the slime molds because it does not exist in all slime molds and was observed in some dinoflagellates, and ascomycetes.

Autoradiography↗

PubMed1983

Uptake and killing of Leishmania mexicana amazonensis amastigotes by human skin fibroblasts.

This paper describes the in vitro infection of human established fibroblast lines by Leishmania mexicana amazonensis amastigotes. Intracellular parasites were located within vacuoles. The proportion of infected cells reached a peak of about 50% on Days 2 or 3, and decreased to almost 0 on Days 6 or 8. Transmission electron microscopy was used to document different stages of the degeneration of L. m. amazonensis amastigotes on Days 4 and 5. The proportion of infected fibroblasts decreased in both the control and the irradiated cells, indicating that dilution of the parasites by cell multiplication was not the main factor in the observed decrease of the infection with time. This conclusion was also supported by studies with fibroblasts prelabelled with 3H thymidine. In these studies, total cell associated radioactivity as well as the total and TCA precipitable radioactivity of the culture medium were similar for infected and non-infected fibroblasts during the course of infection. The abortive infection of fibroblasts by L. m. amazonensis provides a potentially useful model for the study of the relationship between host cells and intracellular parasites.

Cells, Cultured↗