acid-phosphatase and lucifer-yellow

Light and electron microscopy were used to analyse the process of interaction of Streptococcus agalactiae (serotypes Ia, III, and V) with resident and activated mouse peritoneal macrophages. Transmission electron microscopy showed that adherence of the S. agalactiae serotype Ia, but not III and V serotypes, to the surface of activated macrophages triggers the respiratory oxidative burst as revealed by the presence of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase in the phagocytic vacuoles. Fusion of macrophage lysosomes with bacteria-containing phagocytic vacuoles was observed in macrophages treated with Lucifer yellow as well as by localization of acid phosphatase for all serotypes.

Topics: Acid Phosphatase; Animals; Cells, Cultured; Histocytochemistry; Isoquinolines; Lysosomes; Macrophage Activation; Macrophages, Peritoneal; Mice; Microscopy, Confocal; Microscopy, Electron; NADPH Oxidases; Phagosomes; Respiratory Burst; Streptococcus agalactiae

Giardia lamblia, a primitive eukaryotic cell, lacks organelles such as mitochondria, peroxisomes, and a typical Golgi complex and presents a system of vesicles located below the plasma membrane. We used fluorescence and electron microscopy to better characterize the peripheral vesicles. Incubation of living cells with acridine orange showed that the peripheral vesicles correspond to an acidic compartment. Incubation with lucifer yellow, and with horseradish peroxidase, showed labeling of the peripheral vesicles even after several hours. Acid phosphatase was localized in the endoplasmic reticulum and in most of the peripheral vesicles. On the other hand, glucose 6-phosphatase, an endoplasmic reticulum marker, was observed in the endoplasmic reticulum cisternae and in some peripheral vesicles. A similar labeling pattern was observed using the zinc iodide technique, which reveals SH-containing proteins. Three-dimensional reconstruction and electron microscopy tomography of cells stained for acid phosphatase and glucose-6-phosphatase revealed the connection between some vesicles and profiles of the endoplasmic reticulum. Taken together, our observations suggest that trophozoites of G. lamblia present an endosomal-lysosomal system concentrated in a single system, the peripheral vesicles, which may represent an ancient organellar system that later on subdivided into compartments such as early and late endosomes and lysosomes.

Topics: Acid Phosphatase; Acridine Orange; Animals; Biomarkers; Endoplasmic Reticulum; Endosomes; Giardia lamblia; Histocytochemistry; Horseradish Peroxidase; Isoquinolines; Lysosomes; Microscopy, Electron; Microscopy, Fluorescence; Tomography

Helicobacter pylori broth culture supernatants induce eukaryotic cell vacuolation in vitro, a phenomenon that has been attributed to cytotoxic activity. We sought to characterize further the vacuolation of HeLa cells that occurs in response to H pylori culture supernatant. Nascent vacuoles were detectable by electron microscopy after 90 minutes of incubation with H pylori supernatant and were not associated with any identifiable organelle. After 6 days of incubation with H pylori supernatant, vacuoles were membrane-bound structures filled with electron-dense debris, which resembled secondary lysosomes. Acid phosphatase activity was detected within the vacuoles. The vacuoles induced by H pylori supernatant were then compared with vacuoles induced by trimethylamine, a weak base known to induce lysosomal swelling. Neutral red dye rapidly entered the vacuoles induced by either H pylori supernatant or trimethylamine, and both types of vacuoles were reversible. Compared with trimethylamine-induced vacuoles, the vacuoles induced by H pylori supernatant were larger and typically lacked a limiting membrane. In the early stages of formation, vacuoles induced by trimethylamine were labeled by lucifer yellow, a pinocytotic marker, whereas H pylori cytotoxin-induced vacuoles were not. These data suggest that trimethylamine-induced vacuoles arise directly from endocytic compartments, whereas H pylori cytotoxin induces vacuole formation via an autophagic mechanism.

Topics: Acid Phosphatase; Cells, Cultured; Cytotoxins; Fluorescent Dyes; HeLa Cells; Helicobacter pylori; Humans; Isoquinolines; Methylamines; Neutral Red; Staining and Labeling; Vacuoles

Cultured resident murine macrophages are incubated in the continuous presence of the fluorescent endocytic marker Lucifer Yellow and a phorbol ester that activates protein kinase C. Under these steady-state labeling conditions the fluorescent tracer was, for the most part, in a tubular/reticular compartment. Enzyme cytochemical localization of acid phosphatase in the same cells showed essentially a one-to-one correlation between the Lucifer Yellow- and acid phosphatase-containing compartments. Procedures for epifluorescence observation and subsequent enzyme cytochemical examination of the same whole mount cells are described. In addition, chemical fixation methods for the preservation of this labile tubular/reticular compartment are presented.

Topics: Acid Phosphatase; Animals; Biomarkers; Endocytosis; Female; Fluorescent Dyes; Histocytochemistry; Isoquinolines; Macrophages; Mice; Microscopy, Electron; Peritoneal Cavity; Tetradecanoylphorbol Acetate; Tissue Preservation

We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid-phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC-conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in marker-free media before cell fusion to clear any marker from an endosomal compartment. Recipient cells were infected with vesicular stomatitis virus as a fusogen. Donor and recipient cells were co-cultured for 1 or 2 h and then fused by a brief exposure to pH 5. In all cases, extensive exchange of content between donor and recipient cell lysosomes was observed at 37 degrees C. Incubation of cell syncytia at 17 degrees C blocked lysosome/lysosome exchange, although a "priming" process(es) appeared to occur at 17 degrees C. The kinetics of lysosome/lysosome exchange in fusions between cells containing invertase-positive lysosomes and sucrose-positive lysosomes indicated that lysosome/lysosome exchange was as rapid, if not more rapid, than endosome/lysosome exchange. These experiments suggest that in vivo the lysosome is a rapidly intermixing organellar compartment.

Topics: Acid Phosphatase; Animals; beta-Fructofuranosidase; Cell Fusion; Cell Line; Cricetinae; Cricetulus; Dextrans; Female; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Glycoside Hydrolases; Histocytochemistry; Isoquinolines; Kinetics; Lysosomes; Ovary; Pinocytosis; Xanthenes