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PubMed1986

The effect of mannose 6-phosphate on the turnover of the proteoglycans in the extracellular matrix of human fibroblasts.

Human fibroblasts (SL66) were cultured in medium containing 35SO2-4 to label the glycosaminoglycans (GAGs). The cells were then detached from the culture dish to leave radioactively-labeled components of the extracellular matrix, hereafter termed 35S-labeled substrate-attached material. When unlabeled SL66 fibroblasts were plated onto this 35S-labeled substrate-attached material, the cells mediated two distinct events: (a) release of radioactivity from the substrate-attached material into the medium; (b) degradation of certain glycosaminoglycans into radioactive components of very low molecular weight including free radioactive sulfate. In the presence of mannose 6-phosphate, however, the degradation of the substrate-attached material by SL66 cells was partially inhibited. Analyses of this effect in terms of the dose-response curve, saccharide specificity, ammonium chloride sensitivity, and the requirement for cells suggest that both an intracellular compartment and the mannose 6-phosphate receptor that binds lysosomal enzymes at the cell surface may play important roles in the turnover and degradation of certain proteoglycans in substrate-attached material.

Ammonium Chloride↗

PubMed2003

Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells.

Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural beta-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47-57), as well as a similarly labeled 12-residue beta-peptide, beta-(VRR)4, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47-57)) or nucleolus (beta-(VRR)4). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47-57) and beta-(VRR)4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification.

Active Transport, Cell Nucleus↗

PubMed1990

Ammonia induced decrease in glial fibrillary acidic protein in cultured astrocytes.

Previous studies of human hepatic encephalopathy (HE) have shown decreased levels of glial fibrillary acidic protein (GFAP) in Alzheimer type II astrocytes. In view of the important role of ammonia in the pathogenesis of HE, we carried out immunocytochemical and enzyme-linked immunosorbent assay (ELISA) studies on the effect of ammonium chloride (10 mM) on GFAP content in primary astrocyte cultures. There was a 39% loss of GFAP after a four day treatment. There was no fall in total cell protein. Potential mechanisms for this apparent selective loss of GFAP are discussed.

Ammonia↗

PubMed1983

Mechanisms of lysosomal enzyme secretion by human U-937 monocytes.

Lysosomal enzyme secretion was studied in U-937, a mononuclear phagocyte cell line. The secretion of the marker enzyme, beta-N-acetyl-D-glucosaminidase, was low when the cells were challenged with phagocytosable stimuli (asbestos, zymosan, opsonised zymosan). Ammonium chloride and cytochalasin B induced secretion over 24 h of incubation. Phorbol myristate acetate and F-methyl-leucyl-phenylalanine, did not have any effect on secretion. It is suggested that U-937 cells may provide a useful model for the study of mononuclear phagocyte secretory pathways.

Acetylglucosaminidase↗

PubMed1990

Modulation of kidney cell protein degradation by insulin.

Insulin is an established regulator of intracellular proteolysis in several mammalian tissues but little is known about its role in the kidney. The present study was undertaken to determine whether insulin influences protein degradation in isolated rat renal proximal tubules and to investigate its mechanism of action in cultured proximal-like tubular epithelial cells from the opossum kidney. Long-lived protein degradation was determined from the release of carbon 14-labeled valine from previously labeled cellular protein under conditions designed to minimize label reutilization. In isolated tubules, the mean control rate of proteolysis was 2.18% per hour, indicating an appreciable turnover of cellular protein. Insulin (10(-6) mol/L) decreased the rate by 23%. In cultured kidney cells, the rate of protein degradation averaged 1.25% per hour in the presence of serum and 1.68% per hour in its absence, an increase of 34%. High insulin concentrations suppressed this acceleration completely, and physiologic levels inhibited it partially. No evidence was obtained to indicate that insulin action is mediated through stimulation of Na(+)-H+ antiport or through increased amino acid utilization. Ammonium chloride, however, strongly attenuated the serum deprivation response and the inhibitory effect of insulin. The exact mechanisms whereby insulin inhibits proteolysis is not known, but these findings are consistent with an inhibitory action of insulin on the lysosomal pathway.

Ammonium Chloride↗

PubMed1981

Macrophage antimicrobial activity: evidence for participation by lysosomes in the killing of Saccharomyces cerevisiae by normal resident macrophages.

Ammonium chloride added to normal resident mouse peritoneal macrophages reduced the rate of killing of ingested Saccharomyces cerevisiae. It is suggested that this is attributable to the inhibition by ammonia of phagosome-lysosome fusion or of lysosomal protein degradation and that, consequently, lysosomal mechanisms contribute to the normal killing of these yeasts.

Ammonium Chloride↗

PubMed1990

Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptor expression on human neutrophils.

The regulation of monocyte-derived neutrophil chemotactic factor (MDNCF)/interleukin 8 (IL 8) receptor expression by the MDNCF/IL 8 ligand was examined using freshly isolated human peripheral blood neutrophils. MDNCF/IL 8 down-regulated greater than 90% of its own receptor expression within 10 min at 37 degrees C. This down-regulation was associated with internalization of the ligand. The radiolabeled MDNCF/IL 8 molecules after internalization were proteolytically degraded, and trichloroacetic acid-soluble molecules were released into the culture medium starting at 60 min. Lysosomotropic agents could inhibit this degradation of ligand suggesting the involvement of lysosomal enzymes in this proteolytic digestion. MDNCF/IL 8 receptors reappeared on the cell surface within 10 min after removal of free ligands from the culture medium. Cycloheximide did not alter the reappearance of the receptor suggesting that de novo protein synthesis of MDNCF/Il 8 receptors is not involved in this event and that receptors probably recycled. The addition of lysosomotropic agents partially inhibited the reappearance/recycling of the receptors, although none of these agents inhibited the binding of ligand to the surface receptors or ligand internalization. Ammonium chloride reduced the MDNCF/IL 8-induced neutrophil chemotactic response in a dose-dependent fashion. These data suggest that MDNCF/IL 8 receptor expression is dynamically regulated by MDNCF/IL 8 and that the rapid recycling of MDNCF/IL 8 receptors may be essential for the chemotactic response of neutrophils.

Ammonium Chloride↗

PubMed1971

Urea-inorganic phosphate mixtures as prebiotic phosphorylating agents.

Previous attempts to phosphorylate nucleosides by heating with inorganic phosphate succeeeded only when acid phosphates such as Ca(HPO(4))(2) were used. The addition of urea and ammonium chloride to the reaction mixture permits phosphorylation in high yield with neutral or basic phosphates at temperatures in the range of 65 degrees to 100 degrees C. Since the abundant mineral, hydroxylapatite, is a satisfactory substrate for this reaction, we believe that this procedure plausible model for prebiotic phosphorylation.

Alkaline Phosphatase↗

PubMed1998

Constitutive choline transport in Pseudomonas aeruginosa.

Pseudomonas aeruginosa has a choline uptake system which is expressed in bacteria grown in the presence of succinate and ammonium chloride as the carbon and nitrogen source, respectively. This system obeys Michaelis-Menten kinetics with an apparent Km value of 53 microM; its activity is not inhibited by high osmolarities in the medium but is partially inhibited by choline metabolites such as betaine and dimethylglycine.

Ammonium Chloride↗

PubMed2004

Antimalarial quinolines and artemisinin inhibit endocytosis in Plasmodium falciparum.

Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum. By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.

Ammonium Chloride↗

PubMed1984

Stimulation of the release of lysosomal and nonlysosomal granular enzymes from macrophages treated with monensin.

Mouse peritoneal macrophages that had been treated with a monovalent carboxylic ionophore, monensin, selectively secreted lysosomal and nonlysosomal granular enzymes into the medium. When macrophages were incubated with 1 to 10 microM monensin, the release of beta-glucuronidase, beta-hexosaminidase and beta-galactosidase was stimulated time and does dependently. Neither the beta-glucosidase nor acid phosphatase, enzymes bound to the lysosomal membranes, however, were released by monensin. Neutral alpha-glucosidase, shown recently to be localized in nonlysosomal granules of macrophages (15), was released by monensin at concentrations lower than those required for lysosomal enzyme release. Increased release of lysosomal enzymes also took place in a manner similar to that seen with monensin-treated macrophages after treatment of macrophages with weak bases, chloroquine and ammonium chloride. Neutral alpha-glucosidase, however, was not released when chloroquine was present in concentrations that stimulated the release of lysosomal enzymes. The UDP-galactosyltransferase activity of the Golgi apparatus in the macrophages markedly decreased after treatment with low concentration of monensin.

Acid Phosphatase↗

PubMed2004

X-epilectin: a novel epidermal fucolectin regulated by BMP signalling.

This paper reports the cloning and characterisation of a new posterior epidermal marker, X-epilectin, in Xenopus laevis. This gene encodes for a fucolectin, which belongs to the lectin superfamily of carbohydrate binding proteins and specifically binds fucose residues. RT-PCR and in situ hybridisation show that the expression of this gene is switched on during gastrulation and up-regulated during neurula stages and found expressed ubiquitously throughout the epidermis. From tailbud stages, the expression is limited to the dorsal posterior region of the embryo, suggesting that X-epilectin expression is regulated along anteroposterior and dorsoventral gradients during development. In the adult, X-epilectin is mainly expressed in intestinal components, kidney, spinal cord and skin. The effects of growth factors on the regulation of X-epilectin were studied. Change of the fate of animal caps into cement gland or dorsal mesoderm induces a down-regulation of X-epilectin expression in explants treated respectively with ammonium chloride and activin A. We also show that X-epilectin expression is down-regulated by Noggin and tBR and that this effect is inhibited by BMP4 over-expression, suggesting X-epilectin expression is mediated by the BMP signalling pathway.

Activins↗

PubMed1991

Evidence for endocytosis-dependent proteolysis in the generation of soluble truncated nerve growth factor receptors by A875 human melanoma cells.

We have identified nerve growth factor receptor (NGFR) on the cell surface and a truncated nerve growth factor receptor (NGFRt) in the conditioned medium of NGFR-negative cells that have been transfected with either the gene or the cDNA for the full-length receptor. By using cell surface iodination or metabolic labeling of A875 human melanoma cells, coupled with immunoprecipitation, we have determined the half-life of the cell-associated receptor to be approximately 7 h. Concomitant with receptor degradation is the accumulation of NGFRt in the extracellular medium. Approximately one-fifth of the labeled receptor can be recovered as the truncated species. These data support the hypothesis that NGFRt is generated by proteolysis of previously intact receptor. Furthermore, although no specific protease inhibitor assayed could affect this processing, NGFR degradation and truncation were retarded by treatment with: 1) the weak base amines, ammonium chloride or methylamine; 2) the carboxylic ionophore, monensin; or 3) the vacuolar ATPase inhibitor, bafilomycin A1, all agents that dissipate endosomal/lysosomal proton gradients via alternate mechanisms. Incubation of cells at 4 degrees C precluded NGFR degradation and truncation. The presence of ligand did not alter the time course of receptor truncation.

Ammonium Chloride↗

PubMed1982

Renal calculi associated with incomplete distal renal tubular acidosis.

We report 5 cases of renal calculi associated with incomplete type I renal tubular acidosis. The patients presented with a history of recurrent calculi and demonstrated bilateral calculi with corticomedullary junction calcification on x-ray. Stone analysis revealed primarily calcium phosphate stones and metabolic studies demonstrated hypercalciuria, mild depression of serum bicarbonate and inability to lower the pH of a fasting morning urine to less than 5.8. The 24-hour urinary citrate excretion was depressed markedly. The diagnosis was confirmed by an ammonium chloride loading test. Treatment consisted of alkalization of the patients, which corrects the hypercalciuria and increases the urinary citrate level and should decrease the tendency to form stones. The diagnosis of incomplete type I renal tubular acidosis should be considered in patients with recurrent renal calculi.

Acidosis, Renal Tubular↗

PubMed1975

Ammonia intoxication: a hazard during rehabilitation of protein-deprived rats.

The ammonia tolerance of weanling rats raised for 3 to 6 weeks on a protein-deficient diet was compared with that of a group of age-matched control animals raised from weaning on an optimal protein diet. The groups were challenged with a nitrogen load of either ammonium chloride or amino acids. The results of the loading studies indicate that severe protein deprivation increases vulnerability to ammonia poisoning. These observations suggest that ammonia-induced coma may pose a significant hazard during the initial intravenous rehabilitation of malnourished subjects, if replenishment is undertaken too vigorously.

Amino Acids↗

PubMed1996

Effect of pH on in vitro potency of amantadine against Plasmodium falciparum.

Amantadine is a monoprotic weak base that inhibits intraerythrocytic growth in in vitro cultures of Plasmodium falciparum, specifically chloroquine-resistant strains. Changes in the external PH of the medium are expected to result in a shift in the relative proportion of ionized and unionized species of amantadine by virtue of the weak base characteristic of the drug, influencing passage of the drug through the membrane. The ability of amantadine to alkalinize the food vacuole was determined using the accumulation of acridine orange as a vacuolar probe. Drug sensitivity following alteration of the pH gradient was assessed using the hypoxanthine method. Amantadine was able to alkalinize the food vacuole in the millimolar range; however, since its antimalarial activity is in the micromolar range, alkalinization of the food vacuole is not the primary action of the drug. The pH of the medium profoundly influenced susceptibility to chloroquine; the log of the 50% inhibitory concentration (IC50) values were linearly dependent on the external pH in both chloroquine-resistant and chloroquine-sensitive strains. Log IC50 values of amantadine exhibited a linear dependence on external pH in the chloroquine-sensitive strain, but in the chloroquine-resistant strain, a nonlinear parabolic function was found with the minimum IC50 at pH 7.03. Ammonium chloride did not interfere with the antimalarial activity of amantadine. The presence of the amine group on the hydrocarbon cage is essential for the activity of amantadine in Plasmodium falciparum. These results suggest factors in addition to pH gradient are involved in the effect of amantadine, possibly interactions with membrane phospholipids.

Adamantane↗

PubMed2001

Induction of alpha-synuclein aggregation by intracellular nitrative insult.

Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.

Ammonium Chloride↗

PubMed1978

[Synthetic medium for the biosynthesis of tobramycin].

The effect of the sources of carbon and inorganic nitrogen on the biosynthesis of tobrmycin complex was studied. Maltose and ammonium chloride were shown tobe optimal. A synthetic medium for Streptomyces cremeus var. nov. 2242, to tobramyc in producing organism, was elaborated and its optimization was performed using the experiment mathematical modelling, i. e. the method of latin squares. Tobramycin production on the new medium was 6 to 7 times higher than that on the initial medium.

Ammonium Chloride↗