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At least 955 records · Page 53Linked to original sources
PubMed1988

Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol.

The possibility of introducing definite amounts of nonpermeant molecules into electropermeabilized living cells has been approached by quantifying the amounts of Lucifer Yellow (LY; a 457-Da highly fluorescent molecule) and Phytolacca americana (Pokeweed) antiviral protein (PAP; a 30,000-Da ribosome-inactivating protein) retained by the cells after closure of the electric-field-induced transient structures of permeation. Without the electropermeabilization, these two molecules enter the cell only in very small amounts by fluid-phase pinocytosis. Under our experimental conditions, using the NIH 3T3 cells, the intracellular LY concentration can reach a value equivalent to the extracellular concentration and can be regulated by controlling the external concentration. We describe the use of LY in a rapid and efficient test for the determination of the best electrical-shock conditions of other cell lines. After electropermeabilization, PAP is 2 X 10(5) times more cytotoxic. Its toxicity can be detected at external concentrations (10(-11) M) corresponding to less than 10 internalized molecules per electropermeabilized cell. Therefore, after electropermeabilization, the nonpermeant molecules have a direct access to the cytosol and the biological effect of nonpermeant substances can be revealed.

Animals↗
PubMed1992

The effect of staurosporine, a protein kinase inhibitor, on asialoglycoprotein receptor endocytosis.

Receptor-mediated endocytosis via coated pits is modulated by the activity of protein kinases and protein phosphorylation. We examined the effects of the potent protein kinase inhibitor staurosporine (SSP) on endocytosis of the asialoglycoprotein (ASGP) receptor in HepG2 cells. Staurosporine caused a rapid (< 2 min) inhibition of ligand internalization from the cell surface. In contrast the rate of receptor exocytosis from intracellular compartments to the cell surface was not altered (t1/2 = 8 min). This resulted in increased ASGP receptors at the plasma membrane (140% of control) while the total number of receptors per cell was unchanged. Receptor up-regulation was half-maximal at 30 nM SSP. At this concentration staurosporine also inhibited the internalization of iodinated transferrin by HepG2 cells and SK Hep-1 cells, another human hepatoma-derived cell line. Staurosporine was without effect on the non-receptor-mediated uptake of Lucifer yellow by pinocytosis. We investigated the possible involvement of protein kinase C in the inhibitory effects of staurosporine on receptor endocytosis. The active protein kinase C inhibitor H7 did not inhibit ASGP receptor internalization. Furthermore depletion of cellular protein kinase C by overnight incubation with 1 microM phorbol myristate acetate did not abrogate the SSP effect. Together these data suggest that the mechanism of SSP action is independent of the inhibition of protein kinase C. In conclusion staurosporine is a potent and rapid inhibitor of receptor trafficking which is specific for receptor internalization from the plasma membrane.

Alkaloids↗
PubMed1985

Effect of carp gonadotropin (cGTH) and a fraction unabsorbed on concanavalin A-Sepharose obtained from cGTH on vitellogenesis in the hypophysectomized marine teleost Gobius niger.

To investigate whether a differential potency on vitellogenesis exists between the carp gonadotropin (cGTH) and fraction I-cGTH (proteins from cGTH unbound on concanavalin A-Sepharose, which represent 5% of cGTH in weight), hypophysectomized Gobius niger were treated with the two hormonal preparations at the same level. Vitellogenesis was checked for synthesis of vitellogenin and yolk incorporation in the ovary by means of immunological studies and histological techniques (light and electron microscopy). In addition, increased synthesis of vitellogenin was induced by injection of estradiol 17 beta together with each gonadotropin to assess the action of the two hormonal preparations on vitellogenin incorporation. Oogenesis was enhanced by cGTH and fraction I-cGTH, and at the same dose levels both treatments produced a similar pattern of stimulation of vitellogenesis. Vitellogenin was found in all the blood samples of animals treated by the hormones (cGTH and fraction I-cGTH) alone. Vesicles of pinocytosis were detected by electron microscopy up to Stage IIIa of oogenesis. When a high synthesis of vitellogenin was induced by exogeneous estradiol 17 beta injections, the two gonadotropic preparations had similar effects in yolk incorporation. cGTH was not less potent than fraction I-cGTH in these processes even though the cGTH preparation contains only 5% of fraction I-cGTH. The contamination of cGTH by a small amount of material unbound on concanavalin cannot be solely responsible for the vitellogenic activity of cGTH which consists of 95% glycoproteins.

Adsorption↗
PubMed1983

The effect of ion channels on cell membrane motion.

Cell membranes possess ion channels and pumps which allow them to pass electrical currents. In this paper it is shown that the ion channels and pumps should induce electric fields in the area surrounding the membrane. The magnitude of this field is computed. It is shown that when the channels and pumps are separated by a sufficient distance, then the electric fields which they induce will cause the solution which surrounds the membrane to move. This movement of the fluid should cause motion of the membrane. Thus it is demonstrated that under certain conditions the conductance of ions across cell membranes will give rise to cell membrane motion. It is suggested that this phenomenon may play a role in processes such as chemotaxis, cell division, and pinocytosis.

Cell Membrane↗
PubMed1984

Mechanisms governing the relative proportions of thyroxine and 3,5,3'-triiodothyronine in thyroid secretion.

In subjects with normal thyroid function only a minor part of firculating 3,5,3'-triiodothyronine (T3) originates directly from the thyroid; the majority is produced in the peripheral tissues by deiodination of thyroxine (T4). However, T3 of thyroidal origin constitutes a relatively high fraction of the total T3 produced in many patients with thyroid hyperfunction or hypofunction. Such a relatively high T3 content in the secretion of the thyroid could be caused by a low T4/T3 ratio in thyroglobulin. Severe iodine deficiency is a well-known inducer of a low T4/T3 ratio, but a low T4/T3 ratio can also be produced independent of the iodine content. This is seen in in vitro studies of thyroglobulin iodination when small amounts of DIT are added to the incubation mixture and in vivo in TSH-treated animals and in patients with Graves' disease. Another mechanism for high thyroidal secretion of T3 could be an enhanced fractional deiodination of T4 to T3 in the thyroid. In vitro thyroid perfusion studies have shown that the T3 content of thyroid secretions is higher than would be expected from the T4/T3 ratio of thyroid hydrolysate and that the major mechanism is deiodination of T4 to T3. Thyroxine deiodinases are also present in the human thyroid, and the amount of T4 deiodinase is enhanced in the thyroids from patients with medically treated Graves' disease and in the hyperstimulated thyroids of rats. Other factors of possible importance for the mixture of T3 and T4 secreted by the thyroid are a relatively faster liberation of T3 than of T4 from thyroglobulin during partial hydrolysis (this faster release of T3 is probably the mechanism behind the more "rapid" secretion of T3 than of T4), or some kind of thyroid heterogeneity leading to pinocytosis and hydrolysis of thyroglobulin with a lower T4/T3 ratio than that of average thyroglobulin.

Animals↗
PubMed1993

Investigation into the histogenesis of congenital epulis of the newborn.

Five previously unreported cases of congenital epulis of the newborn are presented. All five cases were on the anterior maxillary alveolar ridge. Four were removed at 2 days of age and one at 7 weeks. Light microscopy demonstrated large eosinophilic granular cells within vascular fibrous connective tissue. Immunohistochemical studies revealed a positivity for vimentin and neuron specific enolase. Cytogenetic evaluation performed on one case was normal. Estrogen and progesterone receptors were absent in the one case so studied. Electron microscopy demonstrated tumor cells that were filled with autophagosomes. Cellular organelles were significantly reduced and inversely related to the number of cytoplasmic autophagosomes. Many of the autophagosomes contained collagen precursors. Poorly formed junctional complexes were seen. Occasional tumor cells demonstrated long processes that contained contractile microfilaments, pinocytosis, and areas of exocytosis. These studies suggest the tumor cells represent early mesodermal cells that express pericytic and myofibroblastic features that undergo cytoplasmic autophagocytosis.

Collagen↗
PubMed1976

The fine structure of a parasitic ciliate Terebrospira during ingestion of the exoskeleton of a shrimp Palaemonetes.

The ciliated protozoan, Terebrospira chattoni, invades the exoskeleton of the shrimp, Palaemonetes pugio, eating out long galleries parallel to the surface of the exoskeleton. Solubilization of the exoskeleton occurs around an area of the elaborately infolded surface membrane at the anterior of the organism. Dissolved products of the digestion of the exoskeleton are taken into the body by the formation of coated vesicles at pores in the membrane. The surface membrane that is taken in by pinocytosis is apparently recycled by the introduction into the membrane of organelles implicated in membrane recycling in other ciliates. Acid phosphatase can be demonstrated on the surface membrane as well as in the endocuticle around the organism.

Acid Phosphatase↗
PubMed1979

Apolysis and the turnover of plasma membrane plaques during cuticle formation in an insect.

The apical plasma membranes of Calpodes epidermal cells have small fattened areas or plaques with an extra density upon their cytoplasmic face. The plaques are typically at the tips of microvilli. The are present during the deposition of fibrous cuticle and the cuticulin layer. Since the plaques are close (less than 15nm) to the sites where these kinds of cuticle first appear, they are presumed to have a role in their synthesis and/or deposition and orientation. When fifth stage larval cuticle deposition ceases prior to pupation, the plaques are lost as the area of the apical plasma membrane is reduced. The plaques pass from the surface into pinocytosis vesicles and multivesicular bodies where they are presumably digested. The loss of plaques occurs as the blood level of moulting hormone reaches a peak at the critical period after which the prothoracic glands are no longer needed for pupation. Apolysis or separation of the epidermis from the old cuticle is the stage when plaques are absent, the old ones have been lost but the new ones have yet to form. After the critical period, the epidermis prepared for pupation with a phase of elevated RNA synthesis at the end of which plaques and microvilli reform in time to secrete the new cuticulin layer and later the fibrous cuticle of the pharate pupa. There is a new generation of plaques for each moult and succeeding intermoult and each generation is involved in two kinds of cuticle deposition before involution and redifferentiation.

Animals↗
PubMed1980

The involvement of coated vesicles in the secretion of corticosterone by the zona fasciculata of the rat adrenal cortex.

Following exposure of rats to unpredictable stress there was a marked increase in the number of 'coated' vesicles in contact with or close to the cell membrane of the zona fasciculata cells. The close correlation between the vesicle numbers and the plasma levels of corticosterone led to the hypothesis that the coated vesicles were intimately involved in the secretory process. The use of horseradish peroxidase as a tracer protein confirmed that the coated vesicles were not involved in pinocytosis and the inward movement of materials, this function being performed by a much larger uncoated vesicle. The presence of microtubules associated with the coated vesicles and radiating through the Golgi body region, the site of formation of the vesicles, suggested that they may be involved in the transport of the secretory product to the cell membrane. The use of microtubules inhibitors, colchicine and vinblastine, were found to significantly reduce the plasma steroid response to stress. On the basis of these findings a new secretory mechanism was postulated.

Adrenal Cortex↗
PubMed1980

An ultrastructural study and stereological analysis of the colon wall in the cockroach Periplaneta americana.

The colonic epithelium has been examined for ultrastructural evidence of physiological activity. The cells show extensive folding of the apical plasma membrane, associated with mitochondria and an internal coating of particles about 120 A diameter. Anteriorly many apical infoldings are dilated at the tip to form substantial extracellular spaces up to 0.8 micron wide. Narrow intercellular channels are present, opening to the haemolymph side of the epithelium. Pinocytosis is seen frequently at the basal surface. The surface densities of apical plasma membranes were not significantly different in the posterior mid-gut and colon. Similarly the volume densities of mitochondria were equal in the colonic epithelium and rectal pads, but the surface density of outer mitochondrial membranes was greater in the colon. It is suggested that the colon may absorb organic solutes from the gut lumen.

Animals↗
PubMed1981

Ultrastructural evidence for the endocrine nature of the lateral organs of the cattle tick Boophilus microplus.

The lateral organs of the tick Boophilus microplus were previously thought to have a neurohaemal function, but the present study shows that they consist of glandular cells which contain a rich system of smooth endoplasmic reticulum (SER) and Golgi but no indication of neurosecretory production or release. There is acid phosphatase activity throughout the SER as well as in Golgi and a major function of the latter may be the production of lysosomal enzymes. It is suggested that the organs are endocrine glands and that, in engorged females, may secrete a hormone involved in the control of vitellogenesis. The organs are more active in feeding than in unfed males and a related function could be in control of the development of genital organs or spermatogenesis. Also present in the cells are coated vesicles, lipid droplets and microtubules. Coated vesicles close to Golgi are probably primary lysosomes whereas those near the periphery are shown by ferritin tracer to arise from coated pits. Pinocytosis could be involved in membrane retrieval but, in the absence of evidence for exocytosis, this seems unlikely. It is tentatively proposed that, by analogy with vertebrate and insect endocrine glands, the lateral organs may take up hormone precursor via coated vesicles for storage in lipid droplets and conversion to hormone in the SER. As in other SER-rich endocrine glands, the release mechanism for the hormone or other secretory product of the lateral organs is uncertain. Both the steroid, ecdysone and the terpenoid, juvenile hormone, are discussed as possible candidates for the lateral organ hormones.

Acid Phosphatase↗
PubMed1986

Partitioning of renal zinc between metallothionein and ethylenediaminetetraacetic acid (EDTA) after treatment of rats with Ca(Na)2EDTA.

Calcium disodium edtate (EDTA) is a widely used chelator in the treatment of lead poisoning, although it also mobilizes other trace metals such as zinc. We have found that daily injection of 400 mg EDTA/kg body wt ip to rats results in an increase in the concentration of zinc (Zn) in the kidney when measured by atomic absorption spectroscopy. This increase is not associated with an induction of metallothionein (MT), a protein normally involved in the homeostasis of zinc. In fact, separation of kidney homogenate on a column of Sephadex G-75 demonstrates that virtually no Zn-MT remains in the kidney 24 hr after an EDTA injection, although after 3 days, Zn-MT levels appear to return to normal. Instead, increased renal Zn is found associated with residual EDTA. False high measurements of MT can be obtained from the Cd-saturation/hemoglobin assay due to the presence of residual EDTA in the kidney. Although nearly all of an injected dose of radiolabeled EDTA is rapidly excreted, 0.2 to 0.1% is retained in the kidney 1 day later; this is sufficient to account for the additional Zn. We propose that the small proportion of EDTA incorporated by pinocytosis into lysosomes in the renal cortical cells binds with all free Zn and subsequently depletes the MT concentration. Although the concentration of Zn measured in the kidney is elevated, there is actually less available for Zn-dependent activity.

Animals↗
PubMed1988

Selectivity of methyl mercury effects on cytoskeleton and mitotic progression in cultured cells.

Methyl mercury (MeHg) disrupts microtubules, but effects on other cytoskeleton components are not well studied. Dose-effect relationships were used to determine the selectivity of MeHg effects on vimentin intermediate filaments, actin microfilaments, and microtubules. These were examined in PtK2 cells by immunofluorescence. At 0.5 microM MeHg the number of microtubules was reduced. After 1.0 or 2.0 microM MeHg, microtubules in most cells were disassembled except for a few "stable" microtubules. No effects on vimentin or actin filaments were observed except as secondary effects at concentrations of MeHg that caused extensive microtubule disassembly. Antimitotic effects of MeHg are well known. An assay based on fluid pinocytosis was used here to study kinetics of mitotic progression. HeLa cells were arrested at prometaphase with a lengthening of subsequent stages of mitosis. Progression from prophase to prometaphase was not affected. MeHg treatment also increased the number of micronucleated and multinucleated cells. Drugs specific for actin cause similar effects by blocking cytokinesis but the selective action of MeHg on microtubules argues against this mechanism. Data from both interphase and mitotic cultured cells indicate that MeHg acts selectively on microtubules. It further supports the hypothesis that the mechanism of damage of MeHg is related to its antimicrotubule activity.

Actins↗
PubMed1987

Cutaneous injury by topical T-2 toxin: involvement of microvessels and mast cells.

Topical applications of various doses of T-2 toxin to rats led to delayed skin reactions. Following a dose-dependent latent period of 12-24 hr, there appeared vascular dilation, stasis, edema and mononuclear cell infiltration, with many degranulating mast cells. These signs were earliest and strongest in the subcutis. Epidermal necrosis occurred 1-2 days later and was probably caused secondarily by ischemia, due to microcirculatory failure. Ultrastructurally, endothelial cells of small vessels were the earliest sites of change. While intercellular junctions remained closed and pinocytosis decreased, the cytoplasm contained many ribosomes, vacuoles, and abnormal mitochondria. Another early effect of topical T-2 toxin was an increase in number and degranulation of mast cells, especially in the subcutis. The resemblance of the skin injury to that produced by irradiation is noted.

Animals↗
PubMed1976

Translation of Xenopus liver messenger RNA in Xenopus oocytes: vitellogenin synthesis and conversion to yolk platelet proteins.

Xenopus liver vitellogenin and albumin mRNAs injected into Xenopus oocytes are correctly translated, as shown by specific immunoprecipitation and co-electrophoresis with purified Xenopus vitellogenin (molecular weight 210,000 daltons) and albumin (molecular weight 72,000 daltons). Vitellogenin made in oocytes under the direction of injected liver mRNA is unstable compared to other proteins made on injected messengers (such as albumin and globin) and endogenous oocyte proteins (including actin), the half-life of newly made vitellogenin being about 8 hr. Pulse-chase experiments with 35S-methionine show vitellogenin to be a precursor to yolk platelet lipovitellin (molecular weight 120,000 daltons), while 3H-serine labeling demonstrates conversion to phosvitin (molecular weight 34,000 daltons). In contrast, injected 3H-serine 35S-methionine-labeled Xenopus vitellogenin protein is not converted to yolk platelet proteins and is degraded rather slowly (half-life, 23, 29 hr). Phosphorylation of serine residues in phosvitin can be detected in oocytes injected with 32PO4 or gamma-32P-ATP; thus exogenously derived yolk platelet protein is further modified, or turned over, once it is within the oocyte. Moreover, vitellogenin made in oocytes programed with liver mRNA is phosphorylated. Thus phosphorylation, assembly into yolk platelets, and cleavage are events that do not require vitellogenin supplied by the normal pathoways involved in yolk formation (synthesis and post-translational modification in the liver, transport in the serum, and follicle cell-dependent pinocytosis). Vitellogenin mRNA sediments at about 29S in a sucrose-SDS gradient, while albumin messenger peaks at 16S; both species contain poly(A). These liver mRNAs are functionally stable in oocytes for at least 5 days. Vitellogenin-forming activity, relative to albumin, actin, or total endogenous activity, increases with time, and the final rate of 2-2.5 times the initial rate is only reached 3 days after injection. The potentiation effect probably stems from an increase in the efficiency of translation of vitellogenin mRNA. The availability of homologous mRNAs now permits injected messenger to be used as a valide probe of oocyte function; the biological activity of mRNA from a non-ovarian Xenopus tissue proves that some at least of the translational systems within the Xenopus oocyte are not cell type-specific. Moreover, the whole cell system is eminently suitable for assaying putative translational (and possibly transcriptional) control elements from frog liver.

Albumins↗
PubMed1980

The galactose-specific recognition system of mammalian liver: the route of ligand internalization in rat hepatocytes.

We have used two electron microscopic tracers, asialoorosomucoid covalently coupled to horseradish peroxidase (ASOR-HRP) and lactosaminated ferritin (Lac-Fer), to investigate the internalization of proteins bound by the asialoprotein receptor of rat hepatocytes. Both ligands are cleared rapidly from the circulation of rats, are retarded in their clearance by an excess of ASOR and accumulate principally in the liver. Morphological examination of the livers of rats after injection of the probes confirmed that the hepatocyte is the principal liver cell involved in the clearance of galactose-terminating proteins. Internalization occurred via coated pits and coated vesicles of 1000 A diameter. At 30 sec to 2 min the tracers began to accumulate in a complex arrangement of larger smooth-surfaced vesicles and tubular structures at the sinusoidal periphery of the cell. Fluid phase pinocytosis did not appear to account for any of the uptake into larger vesicles. The particulate tracer, Lac-Fer, was closely apposed to the membrane of coated pits and vesicles, but was found scattered throughout the lumen of the larger vesicles, possibly indicating dissociation of the ligand from its receptor. Although occasional lysosomes were detected cytochemically in the cell periphery, vesicles containing Lac-Fer showed no demonstrable aryl sulfatase activity. At 5 min, the tracers began to appear in Golgi-lysosome regions of the hepatocyte and were present in small vesicles of <2000 A in diameter, larger irregular vesicles and tubules. Serial sectioning indicated that tubular structures in Golgi-lysosome regions were often interconnected to the larger vesicles, but that tubules in the peripheral cytoplasm were only occasionally connected to larger structures. Some of the Lar-Fer-containing vesicles in Golgi-lysosome areas at 15 min after injection were found to contain aryl sulfatase reaction product, indicating fusion with lysosomes.

Animals↗
PubMed1997

Uptake of microparticles by rat visceral yolk sac.

The visceral yolk sac (VYS) is responsible for a major part of the amino acid nutrition of the early post-implantation rat embryo and possibly also at the fetal stage of gestation. The mechanism involves endocytic uptake of proteins by the tissue's epithelial cells followed by intralysosomal digestion to amino acids. The amino acid so generated are used for protein synthesis in both the embryo and the VYS. Previous reports had indicated that the endocytic capacity of the VYS might be limited to exclude larger macromolecules. This study demonstrates that Percoll, which comprises 30-nm silica particles coated with polyvinylpyrrolidone (PVP), is as effectively captured by the 17.5-day rat VYS cultured in vitro as PVP itself. Uptake of 125I-labelled Percoll was progressive with time over 5 h and was inhibited by a low incubation temperature, 2,4-dinitrophenol (50 micrograms/ml), EGTA (5 mM), colchicine (10 micrograms/ml) or cytochalasin B (10 micrograms/ml). After uptake of 125I-labelled Percoll, VYSs released only 20 per cent of their radioactivity when re-incubated in fresh medium for 3 h. These data, and electron micrographs showing Percoll in intracellular vacuoles, are all consistent with uptake by endocytosis. Percoll's rate of uptake by the VYS indicates that, like 125I-labelled PVP, it enters the cell chiefly by fluid-phase pinocytosis. It is concluded that endocytosis by the VYS will efficiently capture even the largest globular proteins, and that previous indications of a relatively low size exclusion reflected the loosely coiled configuration of the synthetic polymers used in the earlier studies.

2,4-Dinitrophenol↗
PubMed1994

Amino acid requirements in the early post-implantation rat conceptus.

Amino acid analysis has been performed on hydrolysates of embryos/fetuses, visceral yolk sacs and ectoplacental cones/placentae from early post-implantation rat conceptuses. The increments in each amino acid between 10.5 and 11.5 days, between 11.5 and 12.5 days, and between 12.5 and 13.5 days, are expressed as percentages of the total amino acid increment. These three profiles are very similar to each other and also strongly resemble the amino acid composition of hydrolysates of rat serum. The results are discussed in the context of the hypothesis that a transudate of plasma is responsible for the amino acid nutrition of the embryo at this stage of gestation, and that inhibition of this pathway can lead to the induction of congenital defects. The results suggest that an inhibition of pinocytosis or lysosomal proteolysis would affect the supply of all protein-derived amino acids to approximately the same extent: there is no indication that the supply of any particular amino acid would be particularly vulnerable.

Amino Acids↗