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[Analysis of the effect of physico-chemical factors on the stability of lead and mercury levels in biological. II. Biological material obtained from exposed individuals. Structural examinations].

In order to identify the structure of sediments produced in the stored urine of persons exposed to lead and mercury compounds, the technique of transmission electron microscopy was used. It was observed that the addition of preserving thiocyanate to urine containing lead may evoke changes in precipitation mechanisms. Sediments of chlorides, among others, ammonium chloride, contain lead.

Drug Stability↗

Effects of various basic compounds on the degradation of formaldehyde-treated 125I-labeled bovine serum albumin in mouse liver lysosomes.

The effect of various basic compounds on degradation of formaldehyde-treated 125I-bovine serum albumin (denatured 125I-BSA) in livers of mice was studied in detail. Five basic compounds (BTTB, chloroquine, methylamine, ammonium chloride, and vinblastine) were tested. All the basic compounds inhibited the degradation of exogenous protein in lysosomes. When p-biphenylmethyl-(dl-tropyl-alpha-tropinium)bromide (BTTB) was used, the most effective inhibition was obtained at the concentration of 3.2 mM. Also, vinblastine, a well-known inhibitor of microtubular function, inhibited the degradation of an exogenous protein to the similar extent as that of chloroquine. The inhibition of protein degradation caused by BTTB closely related to the uptake of BTTB into lysosomes. It is supposed that BTTB accumulates in lysosomes and that it inhibits the hydrolytic enzyme by neutralizing intralysosomal pH. Furthermore, it is supposed that BTTB, a quaternary ammonium compound, becomes a useful tool in the study of protein degradation in lysosomes as well as the typically lysosomotropic compounds (chloroquine, ammonium chloride, and methylamine).

Acid Phosphatase↗

Differentiation-induced changes in the content, secretion, and subcellular distribution of lysosomal cathepsins in the human colon cancer HT-29 cell line.

Enterocyte-like differentiated HT-29 colon carcinoma cells were shown to contain far higher intracellular levels of activity of lysosomal cathepsins B, D, and L than their undifferentiated counterparts. In the latter, inhibition of lysosomal functions by leupeptin or ammonium chloride led to a marked increase in the cell-associated activity of the three cathepsins. High levels of pro-cathepsins B, D, and L were found in the culture media of both HT-29 cell populations. Ammonium chloride and chloroquine, which are known to impair the mannose-6-phosphate-dependent trafficking of lysosomal-targeted proteins, did not increase the secretion of the three cathepsins in either undifferentiated or differentiated cultures of HT-29 cells. Analyses by cell fractionation revealed heterogeneities with regard to the density and the content of lysosomal cathepsins between the two cell populations. Leupeptin induced the accumulation of mature lysosomal cathepsins B and L in light density organelles in undifferentiated HT-29 cells. Altogether, these data demonstrate that (1) the expression and subcellular distribution of cathepsins B, D, and L in HT-29 cells are influenced by their state of enterocytic differentiation, (2) the segregation of lysosomal cathepsins is largely inefficient in this tumor cell line and does not increase upon differentiation, and (3) the mannose-6-phosphate-receptor-dependent pathway plays a minor role in the sorting of the three cathepsins, both in undifferentiated and enterocytic-differentiated HT-29 cells.

Ammonium Chloride↗

Generation of potential in lipid bilayer membranes as a result of proton-transfer reactions in the unstirred layers.

The addition of an uncoupler in the presence of a concentration gradient of weak acids or bases (sodium acetate and ammonium chloride) leads to the generation of a potential on lipid bilayer membranes (LBM) which is positive in sign on the side of the membrane with a high concentration of sodium acetate and negative on the side with a high concentration of ammonium chloride. It is shown that the potential was caused by the pH gradient in the unstirred layers. These effects can be understood in terms of the previously described [Science, 182, 1258 (1973)] model for the transfer of weak acids and bases through LBM. This system described may be useful for quantitation of permeabilities for weak acids and bases through bilayer membranes.

Acetates↗

[Antidotal effect of Stenoloma chusanum(L.) Ching against arsenic and ammonium].

The antidotal effect of four extracts from Stenoloma chusanum on acute poisoning of mice caused by arsenic and ammonium chloride was observed. The result shows: extracts B and C can reduce the mortality of mice remarkably, while extract C can raise the tolerance of mice by raising their LD50 from 31.1 +/- 4.3 mg/kg to 38.2 +/- 5.9 mg/kg. In the case of the mice poisoned by ammonium chloride, the four extracts can also reduce the mortality, but make no significant difference as compared with the control group statistically (P greater than 0.05).

Ammonium Chloride↗

Differences in the ribosomes prepared from lactating and non-lactating bovine mammary gland.

1. Ribosomes prepared from bovine lactating mammary gland are able to synthesize protein, whereas similar preparations from non-lactating glands are not. Washing the ribosome suspensions through a medium containing 0.5m-ammonium chloride enhanced their ability to incorporate phenylalanine into polyphenylalanine. 2. Ribosomes isolated from non-lactating bovine mammary gland, in contrast with those from rat liver and lactating mammary gland, contained significant amounts of extraneous nucleases. These enzymes could be removed by washing with a medium A buffer containing 0.5m-ammonium chloride. 3. Only those ribosomes from functionally active tissues were able to bind polyuridylic acid and phenylalanyl-tRNA.

Ammonium Chloride↗

Rectal ammonia tolerance test in the evaluation of portal circulation in dogs with liver disease.

An ammonia tolerance test (ATT) is described in control dogs and in patients affected by liver disease with or without portosystemic collateral circulation. The administration of a 5 per cent ammonium chloride solution rectally proved to be preferable to oral administration. A statistically significant difference was shown between the results of the ATT in patients with and those without portal shunts, but not between control dogs and patients without portal collateral circulation. A simplified ATT is proposed in which venous ammonia determinations are made before and 20 and 40 minutes after rectal administration of 5 per cent ammonium chloride solution at a dose of 2 ml/kg bodyweight.

Ammonia↗

Amniotic fluid cell culture II. Evaluation of a red blood cell lysis procedure for culture of cells from blood-contaminated amniotic fluid.

Second trimester amniotic fluid samples obtained transabdominally for genetic analysis not infrequently are contaminated with blood. There has been disagreement as to whether blood contamination interferes with the efficiency of culture of amniotic fluid cells for genetic diagnosis. A procedure using ammonium chloride to lyse contaminating red blood cells has been recommended to increase culture success. In this report we document that cultures derived from blood-contaminated amniotic fluids form fewer cell colonies than cultures from clear amniotic fluids. We also show that a recommended procedure using ammonium chloride to lyse red blood cells is ineffectual in improving the efficiency of cell culture, and may be detrimental to successful culturing of cells from bloody amniotic fluids. A hypothesis concerning the mechanism of interference with culture of amniotic fluid cells by contaminating blood is presented and a means of preventing this complication is suggested.

Ammonium Chloride↗

Characterization of lysosomal enzymes from cultured cynomolgus monkey trabecular meshwork cells.

The current study characterizes selected properties of lysosomal enzymes associated with cynomolgus monkey trabecular meshwork (MTM) cells. These proteins may participate in the turnover of macromolecules involved in regulating the aqueous outflow. Intracellular levels of lysosomal enzymes in MTM cells were similar to those found in cultured human fibroblasts. The presence of ammonium chloride increased the secretion rate of certain lysosomal enzymes from 47 to 122% of normal. Column chromatography of the secreted enzymes on the galactose-specific lectin Ricinus communis I demonstrated an increase in the number of accessible galactose residues on lysosomal enzymes secreted in the presence of ammonium chloride. The presence of mannose-6-phosphate receptors on the trabecular meshwork cells was demonstrated by the specific uptake of purified 125I-beta-D-glucosidase. This uptake represented 20% of that observed with cultured human fibroblasts and was inhibited only 50% by the presence of mannose-6-phosphate.

Ammonium Chloride↗

[The penetration of the Marburg virus into eukaryotic cells].

The early stages of infection of Vero-E6 cell culture with Marburg virus, a member of filovirus family, highly pathogenic for man, were studied. Virus multiplication was completely or significantly inhibited by lysosomotropic agents (LTA) of two types: weak base (ammonium chloride) and ionophore monensin. The level of the inhibiting effect was proportional to LTA concentration and was maximal when the drug was introduced into the culture medium before virus inoculation. Complete inhibition of Marburg virus replication in Vero-E6 cells in the presence of 20 (30) mM ammonium chloride ("lysosomotropic blocking") was overcome by a short-time treatment of the cell culture with the virus adsorbed on it using a medium with a weak-acid pH (4.0-5.0). The results are discussed from the point of view of the mode of this virus penetration into eukaryotic cells.

Ammonium Chloride↗

Conductivity and hardness changes in aged compacts.

Batches of sodium, potassium, and ammonium chloride tablets containing no excipients and spray-dried lactose tablets containing 0.5% magnesium stearate were stored at 20 and 76% relative humidity. Electrical resistance and hardness measurements were made within 1 hr after compression and at intervals during a 45-day period. Hardness values of sodium, potassium, and ammonium chloride tablets stored at 20% relative humidity increased from 70 to 200% at 45 days, while conductances decreased 10-fold. Tablets stored at 76% relative humidity showed no increases or slight decreases in hardness with slight increases in conductance. Lactose tablets decreased slightly in hardness with corresponding increases in conductance.

Ammonium Chloride↗

Removal of erythroid cells from umbilical cord blood mononuclear cell preparations using magnetic beads and a monoclonal antibody against glycophorin A.

Umbilical cord blood mononuclear cells isolated by density centrifugation are contaminated by erythrocytes and nucleated erythroid precursors which may exceed 50% of the total cell population, and thus interfere with phenotypic, functional and mRNA analyses. Lysis with hypotonic ammonium chloride can overcome this problem, but interferes with lysosomal function and should be avoided when cell preparations are intended for functional studies. The aim of this study was to develop a technique for removing erythroid cells from cord blood mononuclear cell preparations that would be as effective as ammonium chloride lysis but would not affect cellular function. This paper describes a method using 10F7, a mouse monoclonal antibody against human glycophorin A, and magnetic beads coated with anti-mouse immunoglobulin. The population of cord blood mononuclear cells recovered using this technique was of high purity, good yield and viability, and the cells responded appropriately to stimulation in vitro. To maximise cost-effectiveness, purification with magnetic beads could be performed after two density separations to reduce the quantity of beads required.

Ammonium Chloride↗

Purification of cord blood lymphocytes.

When cord blood is separated using standard methods based on Ficoll-Hypaque, the mononuclear fraction is contaminated with erythrocytes and also with nucleated cells that do not express the leucocyte marker CD45. The contamination with CD45-negative cells can exceed 50%, and will interfere with phenotypic, mRNA or functional analysis. A large proportion of these cells are erythrocyte precursors. The contaminating cells may be removed by lysis with hypotonic ammonium chloride; when the cells are required for studies which are adversely affected by ammonium chloride (such as antigen processing), high purity can be attained by two rounds of density separation.

Cell Separation↗

Vesicular stomatitis virus G protein acquires pH-independent fusion activity during transport in a polarized endometrial cell line.

Entry of vesicular stomatitis virus (VSV), the prototype member of the rhabdovirus family, occurs by receptor-mediated endocytosis. Subsequently, during traversal through the endosomal compartments, the VSV G protein acquires a low-pH-induced fusion-competent form, allowing for fusion of the viral membrane with endosomal and lysosomal membranes. This fusion event releases genomic RNA into the cytoplasm of the cell. Here we provide evidence that the VSV G protein acquires a fusion-competent form during exocytosis in a polarized endometrial cell line, HEC-1A. VSV infection of HEC-1A cells results in high viral yields and giant cell formation. Syncytium formation is blocked in a concentration-dependent manner by treatment with the lysosomotropic weak base ammonium chloride, which raises intravesicular pH. Virus release is somewhat delayed by treatment with ammonium chloride, but virus yields gradually reach those of control cells. In addition, inhibition of vacuolar H(+)-ATPases by treatment with bafilomycin A1 also inhibited cell to cell fusion without altering virus yields. Virions released from infected HEC cells were themselves not fusion competent, since viral entry required an active H(+)-ATPase and a low-pH-induced conformational change in the viral G protein. Thus, the conformation change leading to fusion competence during exocytotic transport is reversible and reverts during or after release of the virion from the infected cell.

Animals↗

Ammonia partitioning between glutamine and urea: interorgan participation in metabolic acidosis.

The distribution of precursor nitrogen between urea and glutamine was studied in control and acidotic rats. Acidosis, either acutely induced with hydrochloric acid or chronically induced with ammonium chloride, resulted in a rise in ammonia and a fall in urinary urea excretion; the percent of urinary nitrogen excreted as ammonia rose from 3.5 +/- 0.4 and 4.9 +/- 0.5 in fed and pair-fed controls to 25.9 +/- 3.9 and 37 +/- 5 in acidosis induced by hydrochloric acid and ammonium chloride. Hepatoportal vein urea concentration differences were significantly reduced, whereas glutamine concentration differences were significantly elevated, consistent with a shift of nitrogen from ureagenesis to glutamine; alanine and ammonia concentration differences were significantly decreased and increased respectively in the acidotic animals, suggesting that former supported urea synthesis whereas ammonia may preferentially support glutamine synthesis. Evidence of a feed-forward involvement of the gut in influencing hepatic nitrogen distribution was suggested by an increased ammonia and decreased alanine release in acidotic rats. Bilateral ureteral ligation was performed on control and acidotic rats to determine the fate of the redirected urinary ammonia. Ammonia did not accumulate in the blood, rather it was initially incorporated into glutamine, elevating the plasma level, and then it was subsequently deposited into urea. The shift of nitrogen back into urea in acidotic animals was confirmed by the greater postligation urea production rates supported by hepatic uptake of both alanine and glutamine. These results are discussed in terms of interorgan participation involving the liver, gut, and muscle in the partitioning of nitrogen between glutamine and urea.

Acidosis, Renal Tubular↗

Adrenocorticotropic hormone in the control of renal tubular hydrogen ion secretion. Balance of non-metabolizable base in ACTH-stimulated weanling rats subject to acid or base loading.

Groups of weanling rats subject to ACTH-stimulation (tetracosactrin, 1 mg kg-1 day-1) were exposed to heavy oral loads of ammonium chloride (approx. 21 mmol kg-1 day-1) or sodium bicarbonate (approx. 40 mmol kg-1 day-1) for 8 days. During loading with sodium bicarbonate, the animals maintained a normal positive external (whole body) balance of non-metabolizable base (NB), excess NB being excreted quantitatively in the urine, whereas loading with ammonium chloride caused a fall in the mean balance of NB from a reference value of 12.1 mmol per 8 days to about zero. However, in the two groups the concentration in plasma of NB rose from the reference value (41 mmol l-1) to the same level (57 and 59 mmol l-1, respectively; P greater than 0.5) despite extreme differences in rates of gastrointestinal NB absorption, despite a significant rise in the rate of endogenous sulphuric acid production and despite the presence of ample chloride in diet and urine. These results indicate that ACTH determines the extracellular concentration of NB at which exchange of NB takes place by influencing, directly or indirectly, the relative rates of renal tubular H- secretion and Cl- reabsorption at any given rate of tubular reabsorption of Na-. Some cybernetic considerations of the disturbance are presented.

Acid-Base Equilibrium↗

An epidermal growth factor-ricin A chain (EGF-RTA)-resistant mutant and an epidermal growth factor-Pseudomonas endotoxin (EGF-PE)-resistant mutant have distinct phenotypes.

H2Oe12 is a mutant HeLa cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of epidermal growth factor (EGF) and the toxic A chain of ricin (RTA). ET-28 is a mutant KB cell line selected for resistance to the toxicity of a chimeric protein conjugate composed of EGF and Pseudomonas exotoxin (PE). In this report we describe the presence or absence, in these mutants, of cross-resistance to the two toxic conjugates and the effects of ammonium chloride, leupeptin, and adenovirus cotreatments on toxin efficacies. ET-28 cells, the EGF-PE-resistant cells, are resistant to both EGF-PE and EGF-RTA. In contrast, H2Oe12 cells, the EGF-RTA-resistant cells, are as sensitive to EGF-PE toxicity as are their parent HeLa cells. Ammonium chloride cotreatment substantially reduces the resistance of H2Oe12 cells to EGF-RTA but has little or no effect on the resistance of ET-28 cells to either EGF-RTA or EGF-PE. Leupeptin has no effect on the toxicity of either chimeric conjugate on any of the four cell lines, effect on the toxicity of either chimeric conjugate on any of the four cell lines, despite its demonstrated ability to inhibit cellular degradation of EGF. In contrast, adenovirus cotreatment enhances the toxicity of EGF-RTA and EGF-PE on all cells tested, and completely nullifies the relative resistance of H2Oe12 and ET-28 cells to these toxic conjugates. H2Oe12 and ET-28 cells appear to be altered in distinct, possibly endosomal, functions.

ADP Ribose Transferases↗