SEARCH · PubMed Health

Results for “AMMONIUM CHLORIDE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 883 records · Page 49Linked to original sources

PubMed2001

Regulation of the catalytic activity of preexisting tyrosinase in black and Caucasian human melanocyte cell cultures.

The activity of tyrosinase, the rate-limiting enzyme for melanin synthesis, is higher in Black skin melanocytes than in melanocytes derived from Caucasian skin. This variation in enzyme activity is not due to differences in tyrosinase abundance or tyrosinase gene activity, but, rather, is due to differences in the catalytic activity of preexisting tyrosinase. In melanocytes, tyrosinase is localized to the membrane of melanosomes and in Caucasian melanocytes the melanosome-bound enzyme is largely inactive. Conversely, in melanosomes of Black melanocytes, tyrosinase has high catalytic activity. Treatment of Caucasian melanocytes with the lysosomotropic compound ammonium chloride or with the ionophores nigericin and monensin results in a rapid and pronounced increase in tyrosinase activity. This increase occurs without any change in tyrosinase abundance, indicating that these compounds are increasing the catalytic activity of preexisting enzyme. Inhibition of the vacuolar proton pump V-ATPase by treatment of Caucasian melanocytes with bafilomycin also increases tyrosinase activity. In contrast to the 10-fold increase in tyrosinase observed in Caucasian melanocytes, neither ammonium chloride, monensin, nigericin, nor bafilomycin is able to increase the already high level of tyrosinase activity present in melanosomes of melanocytes derived from Black skin. Finally, staining of Caucasian melanocytes with the fluorescent weak base acridine orange shows that melanosomes of Caucasian, but not Black, melanocytes are acidic organelles. These data support a model for racial pigmentation that is based on differences in melanosome pH in Black and Caucasian skin types. The models suggests that melanosomes of Caucasian melanocytes are acidic, while those of Black individuals are more neutral. Since tyrosinase is inactive in an acid environment, the enzyme is largely inactive in Caucasian melanosomes but fully active in Black melanosomes.

Acridine Orange↗

PubMed1989

Endoproteolytic activation of Newcastle disease virus fusion proteins requires an intracellular acidic environment.

Effects of weak bases, chloroquine and ammonium chloride, on the intracellular cleavage of the fusion protein precursor (F0) were examined in BHK cells infected with a virulent strain of Newcastle disease virus (NDV). Most of F0 molecules synthesized during a 15-min pulse period were chased out because of cleavage into F1 and F2 within a 60-min chase period in the absence of the weak bases. In contrast, significant amounts of the precursor were found to remain uncleaved when chloroquine or ammonium chloride was present. The uncleaved fusion proteins were incorporated into progeny virions as efficiently as cleaved ones, and about the half of fusion proteins were present as F0 in the virion released by the cells treated with 0.3 mM chloroquine. Taken together with the finding that the trans cisternae of Golgi apparatus and forming secretory vesicles have an acidic pH (K. G. W. Anderson and R. K. Pathak, Cell, 40, 685-643, 1985), the present results suggest that an acidic environment in these compartments is required for intracellular proteolytic activation of NDV fusion proteins.

Ammonium Chloride↗

PubMed1976

Renal function in experimental chronic hydronephrosis. V. Net acid excretion capacity in relation to renal pelvic volume after maximal loading with NH4Cl.

In 12 mongrel dogs intravenous loading with ammonium chloride (4 mmol NH4+/kg body weight) was performed before and 2-9 weeks after unilateral hydronephrosis had been produced by ligation of the ureter over an indwelling catheter. In an attempt to satisfy the demand for adequate, constant, alveolar ventilation a special respirator of the high-frequency positive-pressure ventilation (HFPPV) type was used. The acid-base status of the blood was determined before and after the loading and the acidification capacity of each kidney was assessed by measuring the amount of titratable acid and ammonium ions excreted in the urine. In the hydronephrotic kidney the excretion of both titratable acid and ammonium ions was statistically significantly reduced, both compared with the initial values in session I and compared with the healthy kidney in session II. After additional loading with ammonium chloride in session II, however, both kidneys increased their excretions to approximately similar extents. When the excretion was calculated per ml glomerular filtrate, no statistically significant changes were found between sessions I and II, either for the intact or the hydronephrotic kidney. The renal pelvic volume on the hydronephrotic side was measured and related to the reduction of the capacity of the kidney to excrete titratable acid and ammonium ions. No statistically significant correlation was obtained.

Acid-Base Equilibrium↗

PubMed1992

Some factors influencing the proportion of periplasmic hepatitis B virus pre-S2 antigen in the recombinant yeast Hansenula polymorpha.

A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitis B pre-S2 antigen in the recombinant yeast Hansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that it should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.

Ammonium Chloride↗

PubMed1994

Destabilization of glial fibrillary acidic protein mRNA in astrocytes by ammonia and protection by extracellular ATP.

The expression of the astrocyte-specific intermediate filament protein, glial fibrillary acidic protein (GFAP), is decreased in hepatic encephalopathy and increased in numerous neurological conditions including brain injury. However, little is known about the molecular mechanisms that regulate GFAP expression. Here it is reported that treatment of cultured astrocytes with ammonium chloride reduces GFAP mRNA by up to 85% without inhibiting total RNA synthesis. The effect of NH4Cl was time and dose dependent. The reduction in GFAP mRNA was detected 3 h after initiation of ammonia treatment with a maximum effect observed at 24 h. Significant decreases in GFAP mRNA were observed at 2, 5, and 10 mM NH4Cl. Concurrent treatment with extracellular ATP prevented the loss of GFAP mRNA, possibly by activation of purinergic receptors. In addition, removal of ammonium chloride restored GFAP mRNA to normal levels. Nuclear runoff experiments indicated that NH4Cl did not inhibit GFAP mRNA transcription. Studies using alpha-amanitin, an inhibitor of RNA polymerase II, showed that NH4Cl decreased the stability of GFAP mRNA by approximately 50%. This destabilization of GFAP mRNA may be an important factor in the pathogenesis of hepatic encephalopathy. Because increased GFAP is an important component of reactive gliosis, understanding the mechanisms that destabilize GFAP mRNA may facilitate strategies to minimize the gliosis associated with brain injury.

Adenosine Triphosphate↗

PubMed1995

Effect of urinary alkalinisation and acidification on the tissue distribution of hexachlorophene in rats.

1. Urinary alkalinisation may be helpful in treating acute poisoning with uncouplers of oxidative phosphorylation containing a phenolic hydroxyl (pKa 4-6) or other acidic moiety. 2. We studied the effects of urine alkalinisation and acidification on the tissue distribution of hexachlorophene (HCP, pKa 5.7) in male Sprague Dawley rats (10 rats/group). 3. Ammonium chloride (10 mL kg-1, 2% m/v) or sodium bicarbonate (10 mL kg-1, 2% m/v) were administered by gavage on three occasions over 24 h, prior to a single gavage dose of HCP (180 mg kg-1). Controls received aqueous sodium chloride (10 mL kg-1, 0.9% m/v) followed by either HCP (180 mg kg-1) or vehicle alone. 4. Urine pH, body mass and body temperature were monitored during the study and, at the conclusion of the experiment (12 h post-HCP dose), organ mass (liver, kidney, brain), and plasma, urine and tissue HCP concentrations were measured. 5. No clinical features of toxicity were observed in any group. However, sodium bicarbonate significantly reduced median HCP in liver--median plasma and kidney HCP concentrations were also reduced but not significantly. Conversely, ammonium chloride significantly increased median HCP concentrations in liver and kidney--median plasma HCP was also increased but not significantly. 6. The results provide some support for the hypothesis that blood pH influences the tissue distribution of uncouplers of oxidative phosphorylation containing an acidic moiety. Urinary alkalinisation may be useful in treating acute poisoning with these compounds.

Ammonium Chloride↗

PubMed1975

Xenopus laevis cement gland as an experimental model for embryonic differentiation. II. The competence of embryonic cells.

Explants of the blastocoelic roof of Xenopus laevis embryos were incubated for 6 h in Holtfreter solution containing 10 mM ammonium chloride and 10 mM sodium bicarbonate. After this incubation they were transferred for 5 days to Barth's physiological salt solution. Under these conditions the explants underwent differentiation into cement gland tissue. The ability to produce cement gland was highest when explants were dissected from young gastrulae (stage 10). Explants from younger and older embryos displayed much smaller cement glands. There is no difference in the response of ventral or dorsal portions of the blastocoelic roof of stage-10 embryos under these in vitro conditions involving ammonium chloride stimulation. Although the treatment changed the fate of some ectoblastic tissues, it was unable to force endodermal or mesodermal tissues of the blastoporal lip to differentiate into cement gland. The amount of cement gland in the ectoblastic explants varied according to the egg-batch and on the average accounted for 55 percent of the total tissue. The remaining tissue was undifferentiated. Most of this undifferentiated tissue originates from the adjacent layer of the ectoblast. Therefore it may be estimated that the cement gland accounts for 80 to 100 percent of the volume of the competent superficial layer.

Ammonium Chloride↗

PubMed1976

Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation.

Amino Acid Oxidoreductases↗

PubMed1996

Glycosaminoglycan metabolism in otosclerotic bone cells.

Normal and otosclerotic bone cells were cultured in vitro in serum-free medium to evaluate single glycosaminoglycan (GAG) class synthesis and secretion. Moreover, the degradative process was studied by inhibiting the lysosomal functions through the addition of ammonium chloride to the cultures, an ammine known to inhibit lysosomal degradation by neutralizing organelle activity. Otosclerotic bone cells accumulated a lower amount of GAG both in the cellular and extracellular pool compared to normal ones. The decrease was markedly higher for secreted GAG. Moreover a different pattern of single GAG class distribution was observed in the two cell types considered. In the medium of otosclerotic cells a percentage increase of hyaluronic acid (HA) and dermatan sulphate (DS) and a percentage decrease of heparan sulfate (HS) and chondroitin sulfate (CS) were observed compared to normal bone cells. Ammonium chloride had a lower effect on pathologic than on normal cells, indicating a decrease in the degradative process in otosclerotic bone cells. These results were also confirmed by the experiments on GAG uptake and degradation and by the dosage of enzymatic activity of two exoglycosidases. Since extracellular GAG composition influences bone deposition and mineralization, these data support the hypothesis that otosclerosis is the result of an error in the connective tissue matrix structure.

Acetylglucosaminidase↗

PubMed1991

Assessment of ligand effects in intracellular trafficking of ricin A chain using anti-ricin hybridomas.

Intracellular ricin and immunotoxin trafficking has been difficult to study as only one to two cytosolic ricin A chain (RTA) molecules are sufficient to cause cell death. Previous studies (R.J. Youle and M. Colombatti, J. Biol. Chem., 262: 4676-4882, 1987) using anti-ricin hybridomas identified the secretory pre-Golgi as a critical site for RTA intoxication. We used ricin and RTA immunotoxins constructed with transferrin (TF) or anti-murine TF receptor antibody (RI7/217) to compare patterns of cytotoxicity and intracellular trafficking in anti-ricin hybridomas. Anti-RTA and anti-ricin B chain (RTB) hybridomas bound similar amounts of ricin and secreted comparable amounts of anti-ricin immunoglobulin. Anti-RTA hybridomas were 50- to 500-fold more resistant to ricin than nonsecretory and anti-RTB hybridomas, defining a ricin-resistant phenotype. All hybridomas expressed similar levels of surface TF receptors. RTA immunotoxins were constructed using human TF or RI7/217 and a disulfide linker. In protein synthesis inhibition assays, ricin-resistant hybridomas were manyfold more resistant to RI7/217-RTA than were ricin-sensitive hybridomas. In contrast, all hybridomas were equally sensitive to TF-RTA. Monensin increased ricin cytotoxicity minimally against all hybridomas, but dramatically increased RI7/217-RTA cytotoxicity in ricin-resistant and ricin-sensitive hybridomas in a way that abrogated the ricin-resistant phenotype. In contrast, monensin increased TF-RTA cytotoxicity equally in all hybridomas. Ammonium chloride had little effect on ricin or RI7/217-RTA cytotoxicity, but increased TF-RTA cytotoxicity against all hybridomas. Taken together, these results suggest that RTA molecules mediating cytotoxicity pass through an anti-RTA antibody-containing pre-Golgi compartment when bound to RTB or RI7/217, but not when bound to TF. Monensin abrogates the ricin-resistant phenotype when RTA is linked to RI7/217, but not RTB. This suggests that monensin alters RI7/217-RTA processing proximal to the pre-Golgi and that passage through the pre-Golgi may not be necessary for translocation of RTA to the cytoplasm. Ammonium chloride alters toxin cytotoxicity only when RTA is linked to TF, suggesting that only TF trafficks RTA through an acid-sensitive compartment prior to cytoplasmic translocation. With the addition of potentiating agents, each toxin studied showed a unique cytotoxicity profile against the anti-ricin hybridomas, demonstrating a dominant role of the cell binding ligand in intracellular toxin trafficking.

Ammonium Chloride↗

PubMed1976

The purine nucleotide cycle. A pathway for ammonia production in the rat kidney.

Particle-free extracts prepared from kidney cortex of rat catalyze the formation of ammonia via the purine nucleotide cycle. The cycle generates ammonia and fumarate from aspartate, using catalytic amounts of inosine monophosphate, adenylosuccinate, and adenosine monophosphate. The specific activities of the enzymes of the cycle are 1.27+/-0.27 nmol/mg protein per min (SE) for adenoylosuccinate synthetase, 1.38+/-0.16 for adenylosuccinase, and 44.0+/-3.3 for AMP deaminase. Compared with controls, extracts prepared from kidneys of rats fed ammonium chloride for 2 days show a 60% increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity, and a greater and more rapid synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate. Extracts prepared from kidneys of rats fed a potassium-deficient diet show a twofold increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity. In such extracts the rate of synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate is also increased. These results show that the reactions of the purine nucleotide cycle are present and can operate in extracts of kidney cortex. The operational capacity of the cycle is accelerated by ammonium chloride feeding and potassium depletion, conditions known to increase renal ammonia excretion. Extracts of kidney cortex convert inosine monophosphate to uric acid. This is prevented by addition of allopurinol of 1-pyrophosphoryl ribose 5-phosphate to the reaction mixture.

AMP Deaminase↗

PubMed1982

Ammonia-induced changes in pancreatic hormones and plasma amino acids in patients with liver cirrhosis.

The contribution of hyperammonemia to plasma amino acid imbalance in patients with liver disease was assessed in 10 subjects with chronic hepatitis and in 17 advanced cirrhotics. Insulin, glucagon, and plasma amino acids were determined both in the basal state and 45 min after oral ammonium chloride, at doses used in the ammonia-tolerance test. In cirrhotics, ammonia increased to 3 times basal values, in association with a rise in insulin and, more marked, in glucagon. Aromatic amino acids and free tryptophan further increased, while a significant fall in branched-chain amino acids and glutamate was observed. The increase in ammonia levels strongly correlated with the increase in glucagon (r = 0.707). Two patients, with large esophageal varices, showed signs of disturbed consciousness, in association with a marked rise in ammonia and in the ration of free tryptophan to the sum of neutral amino acids. In patients with chronic hepatitis, whose ammonia levels rose slightly, minor variations in pancreatic glucoregulatory hormones and plasma amino acids were observed, as also happened in 10 healthy subjects following ammonium chloride ingestion. Our data fit with the hypothesis that the plasma amino acid imbalance of cirrhotics may be partly due to ammonia-induced changes in pancreatic hormones.

Adult↗

PubMed2004

Clustering and redistribution of late endocytic compartments in response to Helicobacter pylori vacuolating toxin.

Helicobacter pylori VacA is a secreted protein toxin that may contribute to the pathogenesis of peptic ulcer disease and gastric adenocarcinoma. When added to cultured mammalian cells in the presence of weak bases (e.g., ammonium chloride), VacA induces the formation of large cytoplasmic vacuoles. Here, we report a previously unrecognized capacity of VacA to induce clustering and perinuclear redistribution of late endocytic compartments. In contrast to VacA-induced cell vacuolation, VacA-induced clustering and redistribution of late endocytic compartments are not dependent on the presence of weak bases and are not inhibited by bafilomycin A1. VacA mutant toxins defective in the capacity to form anion-selective membrane channels fail to cause clustering and redistribution. VacA-induced clusters of late endocytic compartments undergo transformation into vacuoles after the addition of ammonium chloride. VacA-induced clustering and redistribution of late endocytic compartments occur in cells expressing wild-type or constitutively active Rab7, but not in cells expressing dominant-negative mutant Rab7. In VacA-treated cells containing clustered late endocytic compartments, overexpression of dominant-negative Rab7 causes reversion to a nonclustered distribution. Redistribution of late endocytic compartments to the perinuclear region requires a functional microtubule cytoskeleton, whereas clustering of these compartments and vacuole formation do not. These data provide evidence that clustering of late endocytic compartments is a critical mechanistic step in the process of VacA-induced cell vacuolation. We speculate that VacA-induced alterations in late endocytic membrane traffic contribute to the capacity of H. pylori to persistently colonize the human gastric mucosa.

Ammonium Chloride↗

PubMed1971

Effects of metabolic acidosis and starvation on the content of intermediary metabolites in rat kidney.

1. Metabolite contents were determined in freeze-clamped kidney from acidotic and starved rats in order to elucidate the rate-controlling steps which are responsible for the acceleration of gluconeogenesis in these situations. 2. In the kidney of rats which were made mildly acidotic by replacing drinking water with 1.5% ammonium chloride for 7 to 10 days (when the plasma bicarbonate concentration was 20mm) the content of phosphoenolpyruvate was increased from the control value of 35 to 63nmol/g and that of 3-phosphoglycerate from 85 to 154nmol/g. 3. Similar but smaller changes in these metabolites occurred in the kidney of starved rats but there were no such changes in the kidney of rats 12h after an infusion of 0.25m-hydrochloric acid, although plasma bicarbonate concentration fell to about 10mm on this treatment. 4. The renal concentration of glucose 6-phosphate was not raised in rats that received ammonium chloride, but was increased in starved and acutely acidotic rats. 5. The concentrations of alpha-oxoglutarate, malate and citrate were less than half the normal value in the kidney of both groups of acidotic rats. These changes can be accounted for on the basis of equilibrium relationships among reversible reactions, particularly as a result of the rise in intracellular ammonia content. A less marked decrease in alpha-oxoglutarate and malate was found in the kidney of starved rats. 6. The renal cortical cytoplasmic oxaloacetate concentration was calculated to be decreased in acidotic and starved rats. 7. These results are discussed in the light of the known enhancement by acidosis and starvation of renal gluconeogenesis. In particular they support the suggestion that the phosphoenolpyruvate carboxykinase reaction is a site of control of gluconeogenesis in kidney in these conditions.

Acidosis↗

PubMed1997

Intracellular alkalinization mobilizes calcium from agonist-sensitive pools in rat lacrimal acinar cells.

1. We have investigated interactions between intracellular pH (pHi) and the intracellular free calcium concentration ([Ca2+]i) in collagenase-isolated rat lacrimal acinar cells. The fluorescent dyes fura-2 and 2',7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF) were used to measure [Ca2+]i and pHi, respectively. 2. Application of the weak base NH4Cl alkalinized the cytosol and caused a dose-dependent increase in [Ca2+]i. Trimethylamine (TMA) also alkalinized the cytosol and increased [Ca2+]i. The increase in [Ca2+]i evoked by NH4Cl or TMA was much smaller than that evoked by the secretory agonist acetylcholine (ACh). 3. Application of NH4Cl also increased [Ca2+]i in cells bathed in Ca(2+)-free medium, indicating that NH4Cl released Ca2+ from an intracellular pool. 4. Ammonium chloride had no effect on [Ca2+]i in cells bathed in Ca(2+)-free medium if agonist-sensitive intracellular Ca2+ pools had been depleted with either ACh or the microsomal Ca(2+)-ATPase inhibitor 2,5-di(tert-butyl)hydroquinone. Treatment of cells with NH4Cl in Ca(2+)-free medium reduced the amount of Ca2+ released by ACh. These results suggest that NH4Cl released Ca2+ from the same intracellular pool released by ACh. 5. Calcium release from the agonist-sensitive pool was also triggered when the cytosol was alkalinized by removing the weak acid acetate. 6. Ammonium chloride caused a modest increase in inositol phosphate production, suggesting that NH4Cl may have released stored Ca2+ via an increase in the intracellular inositol 1,4,5-trisphosphate concentration. 7. The increase in [Ca2+]i evoked by NH4Cl was not sustained even in the presence of extracellular Ca2+. In contrast, when a low dose of ACh was used to evoke intracellular Ca2+ release of similar magnitude, sustained Ca2+ entry was observed. 8. Alkalinizing the cytosol appeared to partially inhibit Ca2+ entry triggered by thapsigargin or by ACh. 9. We suggest that alkalinizing the cytoplasm in unstimulated lacrimal acinar cells can release Ca2+ from the intracellular agonist-sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2+ entry pathway or the mechanism which couples the entry pathway to store depletion.

Acetic Acid↗

PubMed2003

Expression of lysyl oxidase and effect of copper chloride and ammonium tetrathiomolybdate on bovine ovarian follicle granulosa cells cultured in serum-free media.

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the UK dairy herd and, although characterized by reduced or absent oestrus and reduced conception rates, the exact cause remains unknown. The aim of this study was to investigate the expression of mRNA for the copper-dependent enzyme, lysyl oxidase, and the effect of copper and/or copper chelating thiomolybdates on FSH-induced differentiation of bovine granulosa cells cultured in serum-free media. Expression of lysyl oxidase mRNA was investigated using bovine specific primers and RT-PCR on cell lysates obtained from bovine granulosa cells cultured under optimum conditions for 0, 16, 24, 48, 96, 144 and 192 h. The effect of thiomolybdates and copper were investigated by supplementing optimized granulosa cell culture media with ammonium tetrathiomolybdate at 0, 0.1, 1, 10, 100 and 1000 micro g ml(-1), copper chloride at equimolar concentrations (0, 0.0516, 0.516, 5.16, 51.6, 516 micro g ml(-1)) or equimolar combinations of both media. Lysyl oxidase mRNA was expressed by the granulosa cells throughout the 192 h of culture. Thiomolybdate depressed oestradiol production in a dose-dependent manner at doses > 1 micro g ml(-1) and prevented the characteristic clumped appearance of granulosa cells in this serum-free system. Although the supplementation of copper alone had no effect at physiological doses, the use of the equimolar copper and thiomolybdate media ameliorated the effect of tetrathiomolybdates on both oestradiol production and cellular morphology. In conclusion, the results of the present study indicate that lysyl oxidase is expressed by granulosa cells, that thiomolybdates can prevent FSH-induced differentiation of bovine granulosa cells in vitro and that these effects can be reversed by copper supplementation. Overall, these data support the hypothesis that copper-responsive subfertility results from perturbation of the normal pattern of ovulatory follicle growth and development, an effect that may be mediated, at least in part, via lysyl oxidase activity.

Animals↗

PubMed1981

Characterization of hybrid molecules containing fragment A from diphtheria toxin linked to concanavalin A or the binding subunit of ricin toxin.

Earlier we described the synthesis of a toxic, disulfide-linked conjugate between Fragment A from diphtheria toxin (DTA) and the lectin concanavalin A (Con A). Here we report further characterization of this Con A-SS-DTA conjugate and of a nontoxic conjugate containing DTA linked through a disulfide bridge to the binding subunit of ricin toxin, RTB. The Con A-SS-DTA conjugate preparation was heterogeneous, and contained both divalent and tetravalent forms of Con A linked to DTA. The divalent form of the conjugate, which lacked hemagglutination activity, was as toxic as the tetravalent form for 3T3 cells. Consistent with this result, Con A-SS-DTA analogs prepared with the chemically modified dimeric derivatives of Con A, succinyl-Con A and acetyl-Con A, were as toxic as DTA disulfide linked to tetravalent Con A. Two analogs of Con A-SS-DTA containing nonreducible intermolecular linkages between DTA and Con A were at least 1000-fold less toxic than Con A-SS-DTA, although they were indistinguishable from unmodified Con A in binding to 3T3 cells. This suggests that reduction of the disulfide bridge between Con A and DTA may be necessary for conjugate toxicity. Con A-SS-DTA was equally toxic for a variety of cell lines varying in sensitivity to diphtheria toxin, including Vero, CHO, HeLa, and 3T3. The conjugate was also toxic for a diphtheria toxin-resistant variant of V79 cells (V79 Dtxr-3) that has lower affinity than wild type cells for diphtheria toxin. Chloroquine, colchicine, cytochalasin B, and ammonium chloride had no effect on Con A-SS-DTA toxicity for Vero cells, although ammonium chloride and chloroquine inhibited diphtheria toxin action.

Animals↗

PubMed1990

Effect of ammonia on calcium homeostasis in primary astrocyte cultures.

Calcium influx, accumulation and efflux were studied in primary cultures of rat astrocytes treated with ammonium chloride. Treatment of the cells for 3 days with 10 mMN4Cl resulted in a 35% reduction in 45Ca influx. The decrease in calcium influx was dose-dependent between 2 and 10 mM NH4Cl. Short-term (30 min) exposure to ammonia had no effect on calcium influx. Calcium accumulation, as measured by 20-min exposure to 45Ca, decreased after treating cultures with 10 mM NH4Cl for one or 3 days; a greater effect was observed after the 3-day treatment. Studies with lanthanum, an inhibitor of calcium transport, indicated that the effect of ammonia was not due to non-specific leakage of calcium. Calcium efflux was not affected by exposure of the cultures to ammonium chloride. Purinergic-evoked calcium influx and mobilization was not altered by ammonia. While the mechanism(s) of calcium homeostasis affected by long-term hyperammonemia remain to be defined, these results suggest that reduced astrocytic calcium may be related to the pathogenesis of ammonia-related disorders such as hepatic encephalopathy.

Adenosine Triphosphate↗