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D N Skilleter

Publications and source records attributed to D N Skilleter.

17 recordsLinked to original sources
PubMed1992

Characterization of apoptosis in thymocytes isolated from dexamethasone-treated rats.

The induction of apoptosis by glucocorticoids in isolated thymocytes has been studied extensively. However, it is not known whether or not the same changes occur after in vivo glucocorticoid treatment. In order to investigate this, we have studied the changes occurring in thymocytes isolated from rats, from 2-24 hr after a dose of dexamethasone (1 mg/kg), which caused 50% thymic atrophy. Thymocytes were separated into four fractions by isopycnic Percoll gradients. A loss of cells occurred within 2-8 hr, primarily in only one of the two major fractions of normal thymocytes. This loss of normal thymocytes coincided with the appearance of small dense cells with characteristic features of apoptosis including condensed chromatin, increased DNA fragmentation, internucleosomal DNA cleavage and a "hypodiploid" peak on flow cytometric analysis. Striking differences occurred in the cellular composition of the different Percoll fractions with time. Initially (up to 4 hr), the pattern of changes occurring in vivo resembled those found in vitro. However, at later times, the complex fate of apoptotic cells in vivo, such as phagocytosis, are not observed in the in vitro studies.

Animals↗
PubMed1992

Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation.

Apoptosis, a major form of cell death, is characterized by chromatin condensation, a reduction in cell volume and endonuclease cleavage of DNA into oligonucleosomal length fragments. The detection of these fragments by gel electrophoresis, as a DNA ladder, is currently used as the major biochemical index of apoptosis. Here we report that key morphological changes of apoptosis can be dissociated experimentally from the DNA fragmentation produced by endonuclease activity. Internucleosomal cleavage of DNA is thus likely to be a later event in the apoptotic process.

Animals↗
PubMed1992

A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes.

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.

Animals↗
PubMed1991

C-myc expression is maintained during the G1 phase cell cycle block produced by beryllium.

Salts of the toxic metal beryllium have been shown previously to prevent the synthesis of several enzymes essential for DNA replication in proliferating rat hepatic cells in vivo, and to inhibit the division of rat liver-derived BL9L epithelial cells in vitro, specifically during the G1 phase of the cell cycle. The present study shows, however, that exposure of serum-stimulated sub-confluent monolayer cultures of synchronized BL9L cells to inhibitory concentrations of the beryllium salt BeSO4 (50 microM) did not impair expression of the cell proliferation associated nuclear proto-oncogene c-myc. On the contrary, the increased c-myc mRNA levels normally observed during the G1 phase were maintained by continuous exposure of the cells to BeSO4. This response was specific in that other colloid forming metal salts (ZnSO4 and ZrSO4), which did not inhibit cell division, had no affect on c-myc expression, and mRNA levels for the constantly expressed H-2Kb major histocompatibility complex gene (3'Kb) were unaltered by BeSO4 treatment of the cells. The prevention by Be2+ of the down-regulation of c-myc expression in serum-stimulated BL9L cells appears to result from a modulation of the endogenous transcriptional control process for c-myc, which allows a maintained expression of the gene.

Animals↗
PubMed1989

The low uptake of an abrin A-chain immunotoxin by rat hepatic cells in vivo and in vitro.

The therapeutic value of antibody-ricin A-chain conjugates (immunotoxins) as antineoplastic agents is limited by their rapid removal from the circulation, primarily by cells of the liver which take up the immunotoxin through receptor mediated recognition of mannose-containing oligosaccharides in the toxin A-chain. We have therefore examined the uptake by rat hepatic cells of a monoclonal antibody (LICR-LOND Fib 75) conjugate assembled with the ricin related, but carbohydrate free, A-chain of the plant toxin abrin. The abrin A-chain immunotoxin was very poorly taken up in vivo and in vitro by both hepatic parenchymal and non parenchymal cells whereas a comparable conjugate assembled with ricin A-chain was actively accumulated by liver cells particularly the hepatic non-parenchymal cells. Furthermore, the abrin A-chain immunotoxin uptake by non-parenchymal cells in vitro was unaffected by mannose and the immunotoxin bound less readily to liver cells than did the ricin A-chain conjugate, consistent with a proposal that its accumulation by hepatic cells is brought about by endocytosis following non-specific binding or by fluid phase pinocytosis. These results suggest abrin A-chain immunotoxins might be further explored as anti-cancer agents since in some cases they could have an improved therapeutic efficacy over immunotoxins constructed with ricin A-chains.

Abrin↗
PubMed1988

Sensitivity of different phases of the cell cycle to selected hepatotoxins in cultured liver-derived (BL9L) cells.

1. Many toxins are active against dividing cells and cytofluorometric analysis of synchronized dividing liver-derived (BL9L) cells has been employed to study the relative sensitivity of the G1(G0), S and G2/M phases of the cell cycle to selected hepatotoxins. 2. The cytotoxic metal beryllium, which inhibits cell division, caused a specific block at the G1 phase of the cell cycle. 3. Dehydroretronecine, an antimitotic metabolite of the hepatotoxic plant pyrrolizidine alkaloids, retarded progression of cells through the cell cycle with a consistent accumulation at the late S to G2 phase. 4. Exposure of cells to aflatoxin B1-8,9-epoxide, the putative carcinogenic metabolite of the hepatocarcinogen aflatoxin B1, particularly during the early period of S phase, produced morphologically transformed cells.

Aflatoxin B1↗
PubMed1985

Modification of the carbohydrate in ricin with metaperiodate and cyanoborohydride mixtures: effect on binding, uptake and toxicity to parenchymal and non-parenchymal cells of rat liver.

The carbohydrate in the toxic glycoprotein ricin was chemically modified by simultaneous treatment with sodium metaperiodate and sodium cyanoborohydride. This treatment causes oxidative cleavage of the sugar residues and reduction of the aldehyde groups which are formed to primary alcohols. The modification markedly decreased the rapid removal of ricin from the blood by hepatic non-parenchymal cells with only a relatively small increase in accumulation of the toxin by parenchymal cells. Binding, uptake and toxicity of the modified ricin in primary monolayer cultures of hepatic non-parenchymal cells were all decreased to a much greater extent than in parenchymal cells. The results indicate that native ricin binds to non-parenchymal cells by a dual recognition process which involves both interaction of cell receptors with the mannose-containing oligosaccharides of the toxin and binding of ricin to galactose-containing glycoproteins and glycolipids on the cells. However, uptake and toxicity of native ricin in non-parenchymal cells appears to result principally from entry of the toxin through the mannose recognition pathway. By contrast, uptake and toxicity of the expressed essentially through the galactose-recognition route.

Animals↗
PubMed1985

Modification of the carbohydrate in ricin with metaperiodate-cyanoborohydride mixtures. Effects on toxicity and in vivo distribution.

Attempts to target antibody-ricin conjugates (immunotoxins) to designated cell types in vivo may be thwarted by their rapid clearance by hepatic reticuloendothelial cells which have receptors that recognise oligosaccharide side chains on the toxin. The B-chain of ricin contains high mannose type oligosaccharides and the A-chain contains a complex unit (GlcNAc)2-Fuc-Xyl-(Man)4-6, all of which potentially could be recognised by the reticuloendothelial system. Treatment of ricin with a mixture of sodium metaperiodate and sodium cyanoborohydride at pH 3.5 resulted in oxidative cleavage of the carbohydrates and reduction of the aldehyde groups thus formed to primary alcohols. By conducting the modification procedure at acidic pH, both the possibility of Schiff's base formation between the aldehyde groups and amino groups in the protein and the possibility of non-specific oxidation of amino acids were minimised. The extent of the carbohydrate modification depended on the duration of treatment, resulting maximally in the destruction of 13 of the 18 mannose residues and of all xylose and fucose. The toxicity of the modified toxin to cells in culture declined by up to 90% as the carbohydrate was destroyed. This was not due to a reduced ability of the B-chain to bind to cells or of the A-chain to inactivate ribosomes. In contrast to the in vitro results, the toxicity of the modified toxin to mice and rats was elevated by up to fourfold. The modification greatly reduced the clearance of the toxin by non-parenchymal cells in the liver and prevented the damage to hepatic Kupffer and sinusoidal cells and to the red pulp of the spleen that is inflicted by the native toxin. The elevated toxicity to animals appears to be because the modified toxin evades the reticuloendothelial system and persists in the bloodstream for longer periods, thus resulting in lethal damage to vital tissues in the animal at lower dosage. The results suggest that immunotoxins prepared from modified ricin would not be readily cleared by the reticuloendothelial system and so be more effective at killing their target cells.

Amino Acids↗
PubMed1979

Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake.

The relative toxicities of particulate beryllium phosphate, soluble beryllium sulphate and a beryllium sulphosalicylate complex to a rat liver parencymal derived cell line have been examined in culture. Due to the propensity of beryllium salts to form beryllium phosphate in solution the incubation medium used was free of inorganic phosphate. Cell death measured by the loss of cellular lactate dehydrogenase into the medium can be produced within 76 h from beryllium phosphate and beryllium sulphosalicylate or 48 h from beryllium sulphate provided the cells have, irrespective of the form of added beryllium, taken up a minimum of 2--5 nmol Be/10(6) cells. Whilst beryllium phosphate was readily taken up as a particle, beryllium complexed with excess sulphosalicylate was not so markedly accumulated by the cells except possibly by formation of small amounts of beryllium phosphate in the medium as a result of inorganic phosphate lost from the cells. The extent of beryllium uptake from beryllium sulphate quantitatively most resembled that observed for beryllium phosphate but was largely independent of beryllium phosphate formation in the medium and not accompanied by the uptake of the SO42- anion. However, the accumulation of beryllium derived from beryllium sulphate did appear to be associated with the production of a sedimentable from believed most probably to be colloidal beryllium hydroxide. The uptake of all forms of beryllium was temperature sensitive and metabolic inhibitor studies and treatment of the cells with trypsin or neuraminidase supported the view that the distinct behaviour of beryllium derived from beryllium sulphate may be related to the enhanced toxicity of this form both under the conditions used and when administered to experimental animals.

Animals↗
PubMed1977

Oxidative phosphorylation. Halide-dependent and halide-independent effects of triorganotin and trioganolead compounds on mitochondrial functions.

1. Each of five triorganotin and five triorganolead compounds was shown to perturb mithochondrial functions in three different ways. One is dependent and two are independent of Cl- in the medium. 2. Structure-activity relationships for the three interactions are described, and compounds suitable as tools for the separate study of each process are defined. 3. In a Cl- -containing medium trimethyltin, triethyltin, trimethyl-lead, triethyl-lead and tri-n-propyl-lead all produce the same maximum rate of ATP hydrolysis and O2 uptake; this rate is much less than that produced by uncoupling agents such as 2,4-dinitrophenol. 4. Increase in ATP hydrolysis and O2 uptake are measures on energy ultilization when triogranotin and triorganolead compounds bring about an exchange of external C1- for intramitochondrial OH- ions. Possible rate-limiting steps in this process are discussed. 5. In a C1- -containing medium ATP synthesis linked to the oxidation of beta-hydroxybutyrate or reduced cytochrone c is less inhibited by triethyltin or triethyl-lead than is ATP synthesis linked to the oxidation of succinate, pyruvate or L-glutamate. 6. The inhibition of ATP synthesis linked to the oxidation of both beta-hydroxybutyrate and reduced cytochrome c consists of two processes: one is a limited uncoupling and is C1- -dependent and the other is a C1- -independent inhibition of the energy-conservation system. 7. The different sensitivities to inhibition by triethyltin of mitochondrial functions involving the oxidation of beta-hydroxybutyrate and succinate are compared and discussed.

Adenosine Triphosphate↗
PubMed1976

The influence of adenine nucleotides and oxidizable substrates on triethyltin-mediated chloride uptake by rat liver mitochondria in potassium chloride media.

In a 100 mM-KCl medium, pH 6.8, containing ATP increasing concentrations of triethyltin cause an uptake of Cl- into mitochondria with a maximum at 1 muM. This can be inhibited by atractylate or oligomycin, but is virtually unaffected by the presence of rotenone. When the medium contains substrate (pyruvate, beta-hydroxybutyrate or succinate), both in the presence and absence of adenine nucleotides, Cl- uptake is greater with a maximum at 1-10 muM-triethyltin. If substrate oxidation is blocked by respiratory-chain inhibitors the Cl- uptake mediated by triethyltin is inhibited except in the media containing ATP, when the characteristics of Cl- uptake similar to that found in the medium containing ATP alone are observed. Under all conditions tested Cl- uptake is decreased by the presence of 2,4-dinitrophenol. It is concluded that energy from either the oxidation of substrate or the hydrolysis of ATP is associated with the generation of sufficient OH- to enable the triethyltin-mediated Cl-/OH- exchange to occur under the metabolic conditions relevant to this action of triethyltin.

Adenosine Triphosphate↗
PubMed1975

The decrease of mitochondrial substrate uptake caused by trialkyltin and trialkyl-lead compounds in chloride media and its relevance to inhibition of oxidative phosphorylation.

1. In a 100 mM-KCl medium (pH 6.8) containing ATP, triethyltin (1 muM) causes a decrease in the uptake of pyruvate, malate, citrate or beta-hydroxybutyrate by rat liver mitochondria, but no decrease is observed in a 100 mM-KNO3 medium. This response is not modified by the presence of rotenone in the incubation medium. 2. In the KCl medium at least 1 muM-triethyltin is required to cause maximum inhibition of pyruvate uptake. 3. Trimethyltin, tributyltin and the trialkyl-lead analogues at 1 muM, to varying degrees, also cause a decrease in pyruvate uptake by mitochondria only in the KCl medium. 4. Triethyltin stimulates resting respiration of mitochondria with all the substrates tested in the KCl medium but not in the KNO3 medium, yet this stimulation of O2 uptake occurs under conditions when substrate uptake is decreased. 5. In contrast, both O2 uptake during state 3 respiration and ATP synthesis when linked to the oxidation of pyruvate, malate or citrate are strongly inhibited by 1 muM-triethyltin in a KCl medium, but O2 uptake and ATP synthesis during the oxidation of beta-hydroxybutyrate are only slightly affected. In a KNO3 medium O2 uptake and ATP synthesis linked to the oxidation of all substrates are only slightly affected. 6. The relevance of the decrease in substrate uptake by mitochondria caused by triethyltin in a KCl medium to the greater sensitivity of various mitochondrial functions observed in vitro is discussed. It is concluded that decrease of matrix substrate content is probably not the major cause of the greater sensitivity of oxidative phosphorylation to triethyltin in a KCl medium observed previously.

Adenosine Triphosphate↗