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Siderophore and haem iron use by Tritrichomonas foetus.

The ability of the parasitic flagellate Tritrichomonas foetus to use various iron sources for its physiological requirements was studied. The siderophores ferrioxamine B, ferrichrome, triacetylfusarinine, coprogen, enterobactin and pyoverdine sustained growth of the cells under iron-limited conditions, and siderophore iron was incorporated into the major iron protein of T. foetus, ferredoxin. The kinetics of siderophore uptake by the cells indicated that a non-saturable transport is involved, unlike the uptake of a ferrous salt. Siderophore uptake by the cells did not involve extracellular reductive dissociation of the ferric chelates, although T. foetus cells had some ferrireductase activity on ferric citrate. Fluorescent analogues of siderophores were used to show that the siderophores taken up by the cells were in small intracellular vesicles. The fluorescence emission maximum of pyoverdine in these intracellular vesicles shifted from 460 nm to 530 nm, indicating a very acidic environment. The results suggest that a wide range of chemically unrelated siderophores can be taken up non-specifically and efficiently used by T. foetus; the mechanism involved may be pinocytosis and removal of the iron from the siderophores in acidic intracellular vesicles. Haemin also sustained the growth of T. foetus cells under iron-limited conditions. The use of haemin iron by the cells probably involves haem oxygenase, since traces of biliverdin were found in the medium when haemin was the iron source. The iron uptake and ferrireductase activities of the cells do not seem to be regulated by the amounts of iron and copper in the growth medium.

Animals↗

Induction of tolerance to urushiol by epicutaneous application of this hapten on dinitrofluorobenzene-treated skin.

The application of a sensitizing dose of urushiol on a dinitrofluorobenzene (DNFB)-treated skin area significantly diminished the intensity of the urushiol challenge test in guinea pigs. Furthermore, the animals which had been first exposed to urushiol through DNFB-treated skin failed to become sensitized in a second sensitization attempt even when painted on a previously untreated area. This tolerance is hapten-specific and may be reversed by treatment with cyclophosphamide (200 mg/kg) shortly before another contact sensitization attempt to urushiol. In a previous work, we have shown that most of the Langerhans cells present in the DNFB-treated skin area are ATPase-negative and that there exists a link between the membranous ATPase system and the formation of Langerhans cell granules. The latter seem to develop in the course of a mechanism of adsorptive pinocytosis during which ATPase activity "disappears." Thus we suggest that the "unavailability" of ATPase-negative Langerhans cells for adequate processing a second hapten may result from the incapacity of cells lacking their ATPase system to activate the intracellular events that depend on this system and that normally lead to sensitization.

Adenosine Triphosphatases↗

Opening of the blood-brain barrier in Anolis carolinensis. A high voltage electron microscope protein tracer study.

The tight junctions between endothelial cells of capillaries in the forebrain of Anolis carolinensis are a common component of the structural basis for the blood-brain barrier in this reptile. The complexity of these junctions, which is apparent in platinum replicas of freeze-fractured brain capillaries, is unchanged by treatments designed to render the blood-brain barrier of these lizards leaky to horseradish peroxidase. An alternative route for extravasation of horseradish peroxidase, following injection of chameleons with 2.7 mg of D-glucose to render their brain capillaries leaky, is a system of transient cytoplasmic vesicles and vesiculo-tubular channels whose lumina may be open to the luminal or abluminal surface (or both) of the capillary endothelial cell. High voltage electron microscopy (HVEM) of 0.25 and 0.5 micron thick plastic sections of experimental brain capillary endothelium confirmed the existence of vesiculo-tubular conduits. These channels display a sigmoid morphology and are situated in the cytoplasm at angles oblique to the luminal and abluminal surfaces of the endothelium. Occasionally, the channels spanned the entire endothelial wall of the capillary, and in such cases, appeared to connect the lumen with the brain extracellular compartment. HVEM images (including stereo pairs) of the vesiculo-tubular channels show them to have a scalloped, irregular profile consistent with their proposed formation by fusion of pinocytotic vesicles. Also, HVEM examinations of experimental capillaries from peroxidase-treated lizards reveal massive quantities of dense reaction product in cytoplasmic vesicles and vesiculo-tubular membrane compartments of the endothelium, and the complex pleomorphism exhibited by these structures. Observations made in the present study suggest that as a consequence of severe hyperglycaemia, transendothelial channels form in the brain capillaries by fusion of pinocytotic vesicles generated by accelerated pinocytosis at the luminal surface of the endothelium, and subsequently serve as open routes for massive floods of tracer into the central nervous system.

Animals↗

Permeability of Dictyostelium discoideum to fucose and uracil following growth-arrest induced by starvation, hadacidin, and cerulenin.

The uptake of fucose and uracil by Dictyostelium discoideum in either a starvation or drug-induced growth-arrest state was studied. For both nutrients, the uptake was an energy-dependent process. The rate of fucose uptake remained constant for over four hours, while the uracil rate declined after about one hour, in starvation-induced growth-arrest. Under these conditions, fucose was found to be incorporated into membrane-associated glycoproteins and glycolipids, while uracil was incorporated into RNA. The rate of fucose uptake was the same for starvation or hadacidin-induced growth-arrest, but significantly lower for cerulenin-treated cells. In contrast, uracil uptake was slower in hadacidin-treated cells as opposed to starvation or cerulenin-induced growth-arrest cells. Further experiments showed that the incorporation rate of uracil into RNA was faster in hadacidin-treated cells than controls, and the cerulenin-treated cells were slower. The data suggest that the cells arrested in growth by nutrient deprivation retain the capacity to take-up and incorporate nutrients such as fucose and uracil and that pinocytosis is probably the process responsible for uptake.

Antifungal Agents↗

Incorporation and intracellular hydrolysis of 131 I-albumin injected in rat testis.

A method of cell separation by enzymatic attack and filtration was applied after injecting the testes. The results indicated that in the case of the interstitial injections, the fraction rich in interstitial cells captures from 30 to 50 times more RISA than that rich in germinal cells, and from 10 to 20 times more than that rich in Sertoli cells. Following the intratubular injection, radioactivity incorporation was greatly reduced and there were no differences in RISA (radioactivity/mg of protein) among the cell fractions. It was concluded that Sertoli cells do not have in its apical side a phagocytic activity comparable to that of the interstitial macrophages and possibly they incorporate only a small amount of tracer by a process of moderate pinocytosis.

Animals↗

Interactions of surface-active alkyltrimethylammonium salts with the plasma membrane of Acanthamoeba castellanii.

The interactions of three surface-active alkyltrimethylammonium salts (C12-C16) with the plasma membrane of Acanthamoeba castellanii were studied. The surfactants caused a release of K+ from the cells at premicellar concentrations. The lytic effectiveness of the surfactants increased with an increase in the length of the alkyl chain with about an order of magnitude for every two carbon atoms added to the alkyl chain. Binding studies with the C16 homologue revealed that at a concentration corresponding to 50% release of K+ there were about 1.9 x10(10) molecules bound per cell. At prelytic concentrations the surfactants stimulated phagocytosis and pinocytosis. The mode of action of the surfactants on the plasma membrane of Acanthamoeba castellanii is discussed and it is hypothesized that the stimulation of endocytosis is due to a "fluidizing" effect of the surfactants on the lipid bilayer of the plasma membrane.

Amoeba↗

Monocyte functions in diabetes mellitus.

The aim of this study was to investigate the functions of monocytes obtained from 14 patients with diabetes mellitus (DM) compared with those of monocytes from healthy individuals. It was found that the total number of circulating monocytes in the 14 diabetic patients was lower than that from the healthy individuals. Phagocytosis of Candida albicans was decreased in the monocytes from the patients, whereas pinocytosis of acridine and phagocytosis of latex and sheep red blood cells were normal. The chemotactic response towards casein was enhanced. The possible consequences of these findings for the elucidation of concomitant infections in diabetic patients are discussed.

Adult↗

Human blood monocyte function in relation to age.

The human blood monocyte function in relation to age has been studied in 60 healthy persons (age range 24-94 years). No differences between females and males were seen. The number of phagocytized Candida albicans per monocyte decreased with ageing (greater than 70 years). Peripheral blood monocyte count, pinocytosis, phagocytosis of latex beads, chemotactic responsiveness and intracellular killing of Candida albicans were independent of age.

Adult↗

Prevention of protein extravasation in the brain by an anion transport inhibitor in acute experimental hypertension in rats.

Acute experimental hypertension induces protein leakage in the brain. The protein is thought to be, at least to a great extent, transported through the endothelial cells by pinocytosis. An anion transport inhibitor, 4 acetamido-4-isothiocyano-stilbene-2,2'-disulfonic acid, markedly reduced adrenaline or bicuculline-induced leakage of 125IHSA and Evans blue-albumin in all areas of the rat brain. The preventive effect of dexamethasone was less pronounced and no effect was seen of haloperidol.

Acute Disease↗

On the relation between ATP splitting and secretion in the adrenal chromaffin cell: extrusion of ATP (unhydrolysed) during release of catecholamines.

1. Cats' adrenal glands were acutely denervated and perfused, in situ, with Locke's solution.2. When the medullary secretogogue Ba (2-10 mM) was added to the perfusion medium, large amounts of AMP and traces of ATP were found in the venous effluent from the adrenal gland along with the catecholamines. In this respect Ba mimicked the physiological secretogogue, acetylcholine.3. Control perfusions with known concentrations of ATP showed that under such conditions ATP was rapidly broken down within the adrenal vasculature.4. When such intravascular hydrolysis of ATP was suppressed by perfusing with a Ca-free, Mg-free Locke's solution containing 1-2 mM EDTA, catecholamine secretion induced by Ba was accompanied by the discharge of large amounts of ATP but relatively little AMP.5. The ATP that accompanied catecholamine secretion in such circumstances is assumed to derive from the ;heavy' nucleotide-rich chromaffin granules and it is concluded that the mechanism for catecholamine secretion does not depend on hydrolysis of the ATP within these granules.6. The release of ATP (unhydrolysed) supports the hypothesis that the chromaffin granules discharge their contents at the cell surface by the process of ;reverse pinocytosis'.

Adenine Nucleotides↗

Radioautographic and electron-microscopic evidence of rapid uptake of antigen by lymphocytes.

Iodine-125-labeled ferritin molecules were detected by radioautography in the sinuses of the rat popliteal lymph node shortly after injection into the foot pad; they appeared to be taken up by macrophages and phagocytic reticular cells. Electron microscopic examination of the same tissue also revealed ferritin molecules within small lymphocytes as early as 5 minutes after injection. The antigen appeared to be taken up by the process of pinocytosis and was distributed throughout the cytoplasm and nucleus. While the number of ferritin molecules observed in the lymphocyte was much less than that taken into the inacrophage, the observation is significant in understanding the role lymphocytes play during the early phase of antibody response.

Animals↗

Membrane alterations in hemolysis: Internalization of plasmalemma induced by primaquine.

Incubation of normal human erythrocytes with primaquine, a derivative of 8-aminoquinoline, results in internalization of the cell membrane and the formation of intracellular vacuoles. These changes are similar to those observed in other types of cells in pinocytosis. The reduction in surface cell membrane which accompanies internalization of plasmalemma may be generally significant in the destruction of red cells.

Cell Membrane↗

Morphologic effect of dimethyl sulfoxide on the blood-brain barrier.

Dimethyl sulfoxide (DMSO) opens the blood-brain barrier of mice to the enzymatic tracer horseradish peroxidase. A single injection of horseradish peroxidase in 10 to 15 percent DMSO into the tail vein along with 10 to 15 percent DMSO delivered intraperitoneally allowed horseradish peroxidase to fill the extracellular clefts throughout the brain within 2 hours. In the absence of DMSO, peroxidase failed to enter brain parenchyma except through the circumventricular organs. Opening of the blood-brain barrier by DMSO is reversible. Dimethyl sulfoxide stimulated the pinocytosis of horseradish peroxidase by the cerebral endothelium; the peroxidase was then directed to lysosomal dense bodies for degradation. Vesicular transport of horseradish peroxidase from the luminal to the abluminal wall of the endothelial cell was not observed. Dimethyl sulfoxide did not alter the morphology of endothelial cells or brain parenchyma.

Animals↗

Loss of the F-actin binding and vesicle-associated protein comitin leads to a phagocytosis defect.

Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

Actins↗

Macrophage activation in murine African trypanosomiasis.

African trypanosomiasis is accompanied by a profound general immunosuppression in which both suppressive T cells and macrophages (M phi) have been implicated. The present studies define changes in the M phi surface, endocytic and secretory properties, during the infection of mice by Trypanosoma brucei. Peritoneal M phi obtained after the control of the first wave of parasitemia displayed characteristics similar to those activated by intracellular pathogens, such as Mycobacterium bovis bacillus Calmette-Guérin, e.g., the enhanced expression of Ia antigen, decreased M phi-specific antigens, receptors mediating the pinocytosis of mannose-terminal glycoproteins, and an increased ability to secrete plasminogen activator, superoxide anion, and H2O2. Some markers of macrophage activation persisted during the subpatent period before the recurrence of parasitemia, whereas others reverted to normal. Mature T cell function appears not to be essential for M phi activation by T. brucei since the infection of athymic nude mice also induced Ia antigens and plasminogen activator. These studies show that M phi activated by different pathways express common features which may contribute to immune dysfunction observed in trypanosomiasis, as well as in other infections.

Animals↗

Morphogenesis of Bittner virus.

The morphogenesis of Bittner virus (mouse mammary tumor virus) was studied in sectioned mammary tumor cells. Internal components of the virus (type A particles) were seen being assembled in virus factories close to the nucleus and were also seen forming at the plasma membrane. The particles in virus factories became enveloped by budding through the membrane of cytoplasmic vacuoles which were derived from dilated endoplasmic reticulum. Complete virus particles were liberated from these vacuoles by cell lysis. Particles budding at the plasma membrane were released into intercellular spaces. Maturation of enveloped virus occurred after release, but mature internal components were rarely seen in the cytoplasm before envelopment. Direct cell-to-cell transfer of virus by pinocytosis of budding particles by an adjacent cell was observed, and unusual forms of budding virus which participated in this process are illustrated and described. There was evidence that some virus particles contained cytoplasmic constituents, including ribosomes. Certain features of the structure of internal components are discussed in relation to a recently proposed model for the internal component of the mouse leukemia virus. Intracisternal virus-like particles were occasionally seen in tumor cells, but there was no evidence that these structures were developmentally related to Bittner virus.

Animals↗

VAMP3 null mice display normal constitutive, insulin- and exercise-regulated vesicle trafficking.

To investigate the physiological function of the VAMP3 vesicle SNARE (v-SNARE) isoform in the regulation of GLUT4 vesicle trafficking, we generated homozygotic VAMP3 null mice by targeted gene disruption. The VAMP3 null mice had typical growth rate and weight gain, with normal maintenance of fasting serum glucose and insulin levels. Analysis of glucose disposal and insulin sensitivity demonstrated normal insulin and glucose tolerance, with no evidence for insulin resistance. Insulin stimulation of glucose uptake in isolated primary adipocytes was essentially the same for the wild-type and VAMP3 null mice. Similarly, insulin-, hypoxia-, and exercise-stimulated glucose uptake in isolated skeletal muscle did not differ significantly. In addition, other general membrane trafficking events including phagocytosis, pinocytosis, and transferrin receptor recycling were also found to be unaffected in the VAMP3 null mice. Taken together, these data demonstrate that VAMP3 function is not necessary for either regulated GLUT4 translocation or general constitutive membrane recycling.

Adipocytes↗

Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

Animals↗