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The fine structure of the interhemal membrane of the rat chorioallantoic placenta during prolonged pregnancy.

In placentas from viable rat fetuses which surgically had been retained beyond term in lactating animals for as long as four days, the fine structure of much of the interhemal membrane was quite like that of full-term placentas. Moreover, contrary to what has been reported previously (Jollie, '64a), in retained placentas which were autopsied at term there were few changes in the four cytoplasmic layers of the membrane which could be interpreted as degenerative. With prolongation of the placenta beyond normal delivery the number of pericytes which were associated with the allantoic endothelium increased; the basal lamina separating the endothelium from trophoblast III progressively became more focally split; and the apposed plasma membranes between this latter layer and trophoblast II appeared to dissociate. Trophoblast II became regionally divided in its cytoplasmic organization, its more superficial zone becoming highly infolded and/or vacuolar. As a consequence, the intertrophoblastic space between layers I and II became more extensive and complex. Within this space, a flocculent material appeared; and there was ultrastructural evidence of massive pinocytosis from the space by trophoblast II. With increased prolongation of gestation, a variety of lysosome-like inclusions appeared within this layer of the membrane.

Allantois↗

Gold-induced changes in the morphology and functional capabilities of human monocytes.

The capacity of gold compounds to induce morphologic changes and alterations in the functional activity of human mononuclear phagocytes (M phi) in vitro was examined. Human peripheral blood mononuclear cells were incubated with gold sodium thiomalate (25 microgram/ml) for 96 hours. As a result, M phi developed electron dense precipitates within phagolysosomes, as well as marked dilatation of these organelles. Gold incubation also altered a number of M phi functions. While viability and adherence were unaffected, the capacity to spread on surfaces was diminished. Pinocytosis of soluble proteins and phagocytosis of opsonized sheep erythrocytes were impaired, but Fc mediated particle binding was not. These data indicate that gold can alter certain functional activities of M phi and support the idea that the major action of gold in rheumatoid arthritis results from its capacity to alter M phi function.

Cell Adhesion↗

Activation of rat synovium by iron.

We observed a consistent sequence of activation changes in rat knee synovia following a single intravenous injection of sterile ferric citrate at a dosage sufficient to cause a transient saturation of transferrin. The initial alteration, appreciated 2 hours postinjection, consisted of an expansion in the lumen of synovial lining and subsynovial tissue due to the hypervascularity and increased cellularity was noted, along with peaks in the mitotic activity of synovial cells (fourfold over baseline) and pinocytosis by endothelial cells. During the period of 8-24 hours, mature collagen appeared in the space between pericyte layers and between pericytes and endothelial cells, and 2-9-fold increases in ferritin +, W3/13+, W3/25+, Ox8+, and Ox26+ mononuclear cells occurred. In contrast to the action on synovial fibroblasts, iron injection did not affect the mitotic activity of fibroblasts within the serosa of the small intestine. These findings demonstrate that, under experimental conditions, iron can readily induce changes in the synovium that are reminiscent of early events occurring in response to antigen.

Animals↗

Constitutive macropinocytosis allows TAP-dependent major histocompatibility complex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells.

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage-colony-stimulating factor have potent T cell-stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II-restricted responses, and more recently using peptide-loaded and protein-pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell-for-cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen-specific cytotoxic T lymphocyte response following adoptive transfer in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Electron microscopic studies of Dane particles in hepatocytes with special reference to intracellular development of Dane particles and their relation with HBeAg in serum.

Electron microscopic observations in 30 cases of HBsAg positive liver disease and 12 asymptomatic carriers of HBsAg suggested the following mechanism of intracellular development of Dane particles: core particles migrated from the nucleus into the cytoplasm through the nuclear pores. Intracytoplasmic core particles protruded into the cisternae of endoplasmic reticulum by budding the outer coat of Dane particles being derived from the membrane of endoplasmic reticulum. Release of Dane particles into the blood stream by reversed pinocytosis was suggested by the finding of submembranous localization of endoplasmic reticulum containing these particles. No budding from the cell surface of the hepatocytes was encountered. Dane particles in the hepatocytes were detected in 14 of 15 cases positive for serum HBeAg while no particles were seen in 27 HBeAg negative cases, thus suggesting that serum HBeAg reflected ongoing replication of hepatitis B virus in the hepatocytes.

Endoplasmic Reticulum↗

Differentiation in Acanthamoeba castellanii is induced by specific monoclonal antibodies.

Monoclonal antibodies that bind a large molecular weight plasma membrane protein of Acanthamoeba castellanii cause the cells to differentiate. A different monoclonal antibody that binds specifically to the major plasma membrane protein has no effect upon cell division or differentiation. The induction of differentiation by the monoclonal antibodies requires a bivalent attachment, more than a single binding cycle of the antibody to the plasma membrane protein, does not require cell-cell contact, and appears to be mediated by an inhibition of pinocytosis. These results suggest one of two alternatives: either this free living amoeba possesses a cell surface receptor that serves to initiate the differentiation process when stimulated, or the specific plasma membrane antigen for the differentiation-inducing monoclonal antibodies is an essential component of the pinocytotic mechanism. While it seems more likely on the basis of available evidence that we are observing the biological effects of a cell surface receptor, either of the two alternative circumstances open up investigative areas of large significance.

Amoeba↗

Antibody to the phosphomannosyl receptor inhibits recycling of receptor in fibroblasts.

The 215-kd phosphomannosyl receptor is involved in the transport of newly synthesized acid hydrolases to lysosomes and also mediates the pinocytosis of lysosomal enzymes by fibroblasts in culture. Recycling of receptors to the sorting sites is an integral part of both these processes. In this report, we describe the inhibition in human fibroblasts of both functions of the phosphomannosyl receptor by a rabbit antiserum to the bovine liver receptor. This inhibition cannot be completely accounted for by inhibition of ligand-receptor interaction. Rather the antibody appears to cross-link receptors and cause a removal of receptors from the sorting sites (plasma membrane and Golgi apparatus) and their accumulation in a compartment from which they do not recycle. Removal of receptors from the recycling pool by antibody is irreversible, and return of receptors requires synthesis of new protein. Degradation of "trapped receptors" is enhanced (t1/2 = 7.5 hr), but much more gradual than their removal from the functional receptor pool (t1/2 = 30 min).

Animals↗

Growth, biochemistry, and morphology of isolated rabbit aortic smooth muscle cells maintained in the presence or absence of serum.

Arterial smooth muscle cells undergo marked biochemical and morphological changes upon culturing. We have studied the time course of these changes in smooth muscle cells isolated from normal rabbit aortas by enzymic digestion and then maintained in Dulbecco's modified Eagle's medium with or without 10% rabbit serum. Subcultured smooth muscle cells were also examined. Isolated cells cultured in the presence of serum multiply rapidly and by 9 days exhibit features typical of subcultured cells including multilayered growth, elevated marker enzyme activities of subcellular organelles, and proliferation of organelles. In contrast, isolated cells cultured in the absence of serum remain quiescent, as indicated by the low level (greater than 10%) of 3H-thymidine incorporation into nuclei and constant DNA content of the cultures, These cells spread slowly to form a monolayer of randomly oriented cells and they retain differentiated morphological features. Their enzyme activities remain at the levels of those of freshly isolated cells initially, but by 5 days some enzyme activities increase, in particular those of the acid hydrolases and catalase. Rates of pinocytosis and protein synthesis in these cells are comparable to those of cells maintained in serum-supplemented medium for the same period, but are significantly less than those measured in subcultured cells. Within 5 days, morphological alterations in the serum-deprived cells occur including the presence of increased numbers of lysosomes. Quiescent cultures of enzymically isolated cells may be a useful tool for short-term biochemical and physiological studies of differentiated arterial smooth muscle cells.

Animals↗

Alpha 1-adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: evidence for compartmentalization of quin-2 and fura-2.

This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to alpha 1-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10(-5) M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180-300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+-indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells "scrape-loaded" with fura-2 revealed a four-fold increase in [Ca2+]i concentration following stimulation with phenylephrine.

Aminoquinolines↗

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages.

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.

Animals↗

Assessment of thyroid function in adult medaka (Oryzias latipes) and juvenile rainbow trout (Oncorhynchus mykiss) using immunostaining methods.

The effect of injected bovine TSH on the pattern of anti-T(4) and anti-T(3) immunostaining of the thyroid tissue was examined in rainbow trout (Oncorhynchus mykiss) and medaka (Oryzias latipes) to determine if the previously reported immunostaining of the cytoplasm of thyrocytes is due to the presence of colloid pinocytotic vesicles and is thus indicative of thyroid hormone release. We hypothesized that the number of immunostained thyrocytes should increase following a TSH challenge, and this should parallel other indicators of increased thyroid activity. In medaka, immunostained thyrocytes were only found following the TSH challenge, and were most marked after 24 to 72 hours; the immunostaining was associated with large colloid-filled cytoplasmic vesicles. In trout, the number and staining intensity of immunostained thyrocytes were increased after the TSH challenge; the immunostaining was present throughout the cytoplasm of the thyrocytes. These findings support the working hypothesis that the immunostaining of the thyrocytes is associated with the pinocytosis of thyroglobulin by the thyrocytes in parallel with an increase in release of thyroid hormone, and that this investigational approach provides a reliable indicator of thyroid hormone release activity.

Animals↗

Adenovirus endocytosis.

Pathogen entry into cells occurs by direct penetration of the plasma membrane, clathrin-mediated endocytosis, caveolar endocytosis, pinocytosis or macropinocytosis. For a particular agent, the infectious pathways are typically restricted, reflecting a tight relationship with the host. Here, we survey the uptake process of human adenovirus (Ad) type 2 and 5 and integrate it into the cell biology of endocytosis. Ad2 and Ad5 naturally infect respiratory epithelial cells. They bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors and this triggers a variety of cell responses, including endocytosis. Ad2/Ad5 endocytosis is clathrin-mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process is induced simultaneously with viral uptake, macropinocytosis. Together, these pathways are associated with viral infection. Macropinocytosis requires integrins, F-actin, protein kinase C and small G-proteins of the Rho family, but not dynamin. Macropinocytosis per se is not required for viral uptake into epithelial cells, but it appears to be a productive entry pathway of Ad artificially targeted to the high-affinity Fcgamma receptor CD64 of hematopoietic cells lacking CAR. In epithelial and hematopoietic cells, the macropinosomal contents are released to the cytosol. This requires viral signalling from the surface and coincides with particle escape from endosomes and infection. It emerges that incoming Ad2 and Ad5 distinctly modulate the endocytic trafficking and disrupt selective cellular compartments. These features can be exploited for effective artificial targeting of Ad vectors to cell types of interest.

Adenoviridae↗

[Electronoptical studies of the effect of 1-[p-(methylnitrosamino)-benzylidenamino]-adamantane on the fowl plague virus (FPV) in cell culture].

The adamantanamine derivative 1-[p-(methylnitrosamino)-benzylidenamino]-adamantane (MBAA) at a concentration of 40 microgram/ml demonstrated no effect on adsorption of fowl plague virus (FPV) on chick embryonal cells. The penetration of the virions took place by means of pinocytosis. In the final stages of penetration the virions became gradually disintegrated. Under the influence of MBAA, after break-down of the membrane of pinocytic vesicles a swollen part of the virus core remained in cytoplasm. The morphologically visible replication stages were completely blocked by MBAA. From these results it was concluded that the antiviral action of MBAA most probably depends on a block of virus replication between the final stages of the penetration process and the beginning of production of virus specific structural antigens.

Adamantane↗

Effect of the intracellular localization of a Gd-based imaging probe on the relaxation enhancement of water protons.

Gd-HPDO3A has been internalized into rat hepatocarcinoma cells in the cytoplasm (by electroporation) or in intracellular vesicles (by pinocytosis), respectively. In the former case, the observed relaxation rates are likely dependent upon the amount of internalized paramagnetic complex, whereas in the latter case the relaxation enhancement is "quenched" to a plateau value (about 3 s(-1)) when the entrapped amount of Gd-chelate is higher than 1 x 10(10) Gd/cell. The observed behavior has been accounted in terms of a theoretical treatment based on equations formally derived by Labadie et al. (J Magn Reson B 1994;105:99-102). On this basis, entrapment into intracellular vesicles has been treated as a three-site water exchange (extracellular/cytoplasm/vesicle compartments), whereas the cell pellets containing the paramagnetic agent spread out in the cytoplasm can be analyzed by a two-site exchange system.

Animals↗

Effects of simple lipids on macrophages in vitro.

Tricaprin increase the rate of spreading of macrophages on glass. The treated cells showed increased pinocytosis and are avidly phagocytic. Cholesterol oleate delays the onset of phagocytosis which however proceeds efficiently once it has begun. Ethyl palmitate induces abnormal movement and the formation of unusual club shaped pseudopodia; ethyl palmitate prevents the ingestion of antibody coated erythrocytes and it is postulated that this substance interferes with the syntheses of IgG receptors.

Animals↗

Facsimile epithelioid cells obtained from stimulated peritoneal macrophages and their secretory activity in vitro.

Mouse peritoneal macrophages collected after exposure to new born calf serum in vivo were found to have some of the morphological and ultrastructural features attributed to epithelioid cells. These features were accentuated by short term culture in vivo or in vitro. Three accepted varieties of epithelioid cell were reproduced, i.e., those with predominant rough ER, those with predominant smooth vesicles and the plasmacytoid variety. Cells resembling fibroblasts were not seen, nor were lymphoid cells. The facsimile epithelioid cells had considerable secretory activity for a range of macrophage enzymes. They retained the phagocytic capacity and the surface receptors of macrophages but to a reduced extent. It is suggested that epithelioid cells are a form of stimulated macrophage, especially effective in enzyme secretion and liable to appear after excitation of the cell membrane by pinocytosis rather than phagocytosis.

Animals↗

Autoreactive MHC-restricted cytotoxic cells in BALB/c mice after novel immunisation with a conserved mammalian autoantigen.

We investigated whether a novel immunisation scheme using an endogenous protein could stimulate an autoreactive cytolytic response. The protein selected was porcine dihydrolipoamide dehydrogenase, the E3 component of the mitochondrial 2-OADC enzyme family, because it is structurally conserved in mammals and ubiquitously expressed. Recombinant insulin was used as an alternative antigen. Female BALB/c mice were injected with adjuvant-free syngeneic lymphoid cells that had been exposed to E3 in hypertonic medium to facilitate its pinocytosis and were given two booster injections. Effector lymphoid cells from immunised mice were cultured in vitro with irradiated syngeneic cells that had been treated with hypertonic medium, either with or without antigen. Cytolytic effector cells were detected that lysed isogeneic and not allogeneic target cells, but only from mice immunised with E3. This experimental system provides a new model for the early stages of the development of autoimmunity.

Animals↗

Effect of transferrin on the degradation of glycoproteins bearing a hybrid or high-mannose glycan by alveolar macrophages.

A subfraction of hen ovalbumin and a special form of rat transferrin, possessing a hybrid glycan, were studied with respect to their binding to, and degradation by macrophages isolated from the rat lung. Both ligands were found to be degraded partly through the mannose receptor pathway and partly by another mechanism, presumably adsorptive pinocytosis. Catabolism of both proteins was markedly increased by adding standard (i.e., normally glycosylated) transferrin from various species to the medium. This increase was not diminished, or even augmented, when Ca2+ was depleted in the medium, thus implying involvement of the pinocytic pathway rather than the mannose receptor. Binding to the cell surface of both ligands was altered in the presence of transferrin. A hypothesis is advanced to suggest that when transferrin is bound to a component of macrophage plasmalemma, thought to be the glycosaminoglycan of heparan sulfate, its conformation may change in such a way that it attracts other proteins. Protein molecules temporarily captured by adsorbed transferrin would then be taken up by a "piggyback" process and degraded.

Animals↗