Levels of malondialdehyde production in rat liver following loading and unloading with iron

Rats were given daily injections of an iron sorbitol citric acid complex in a total dose of 50 mg Fe3+/100 g of body weight and either killed immediately after iron loading, or investigated 2 months later. Among the latter animals, one group was subjected to weekly phlebotomies in order to mobilize iron from the stores, while another group was not further treated. Quantitation of iron and malondialdehyde production was performed on homogenates of liver, kidney and spleen from controls and rats in the different experimental groups, and the distribution of iron in granular form was studied in the livers by means of electron microscopy. The results showed substantially increased amounts of iron in the organs studied after iron-loading and also augmented malondialdehyde production in the liver and kidney (but not in the spleen). A decreased malondialdehyde production was recorded two months after iron-loading in the kidney and spleen of non-bled animals; this decrease was exaggerated in the same organs from bled animals. The production of malondialdehyde as well as the iron content in the livers of both bled and non-bled rats 2 months after iron loading was higher than in the controls. The evidence obtained suggested that the accumulation of iron in the liver was causally related to increased lipid peroxidation. Judging from the morphological appearances this change did not result in cell damage, the only pertinent morphologic alteration being the occurrence of iron particles in the lysosomal vacuome and the cell sap.     

The effect of serum and experimental variables on the transferrin and reticulocyte interaction.

The transfer of iron from transferrin to the developing erythrocyte is a research area of high interest and considerable controversy. We have found that the results of transferrin-reticulocyte incubation studies are quite sensitive to the experimental procedures that are utilized. Reticulocytosis has been induced in rabbits by phelbotomy and phenylhydrazine injections. While the latter gives a higher reticulocyte count, the cells appear to exhibit an altered transferrin-membrane interaction. Transferrin has been iodinated by published methods utilizing chloramine-T and molecular iodine. The iodotransferrin products exhibit the same iron donation ability, however, evidence was found that the chloramine-T treatment leads to a nonspecific binding of transferrin to the reticulocyte. The means of saturating transferrin with 59Fe is also of prime importance. Fe(NH4)2(SO4)2 and especially FeCl3 were found to yield nonspecifically bound iron when added to transferrin or serum. This artifact was reflected in an altered transferrin-reticulocyte interaction. Using what we believe to be optimal conditions, the effect of serum on the transferrin-reticulocyte system was re-examined. The results clearly indicated an enhancement of iron uptake by reticulocytes in the presence of serum, as well as an accelerated incorporation of iron by the cytoplasmic fraction.     

Studies on the rat liver following iron overload: An analysis of iron and lysosomal enzymes in isolated parenchymal and non-parenchymal cells

Iron overload is known to affect the liver. In order to study the effect of iron on various liver cellular and subcellular compartments and the alterations due to mobilization of iron, an experimental model has been developed previously. In this study iron stores in parenchymal and non-parenchymal cells have been investigated during iron loading and unloading. Following completion of the experimental procedures, liver cells were isolated by means of collagenase perfusion (parenchymal cells) and pronase treatment (nonparenchymal cells). It was found that iron overload did not result in significantly increased levels of three lysosomal enzymes, and that the enzyme activities were not altered as iron was mobilized. Iron stores were localized largely in parenchymal cells, and these stores decreased after cessation of iron loading. The iron content was further lowered if the animals were bled. The non-parenchymal cells of the liver initially stored a relatively small part of the administered iron but this increased in the two months following iron loading. On the other hand if the animals were bled there was a pronounced decrease in iron content of these cells as well as in parenchymal cells. It is concluded that iron overload does not affect lysosomal enzymes and that iron stores in both parenchymal and non-parenchymal cells can be mobilized in response to increased demand.     

The effect of serum and experimental variables on the transferrin and reticulocyte interaction

The transfer of iron from transferrin to the developing erythrocyte is a research area of high interest and considerable controversy. We have found that the results of transferrin-reticulocyte incubation studies are quite sensitive to the experiemental procedures that are utilized. Reticulocytosis has been induced in rabbits by phelbotomy and phenylhydrazine inhections. While the latter gives a higher reticulocyte count, the cells appear to exhibit an altered transferrin-membrane interaction. Transferrin has been iodinated by published methods utilizing chloramine-T and molecular iodine. The iodotransferrin products exhibit the same iron donation ability, however, evidence was found that the chloramine-T treatment leads to a nonspecific binding of transferrin to the reticulocyte. The means of saturating transferrin with ⁵⁹Fe is also of prime importance. Fe(NH₄)₂ (SO₄)₂ and especially FeCl₃ were found to yield non-specifically bound iron when added to transferrin or serum. This artifact was reflected in an altered transferrin-reticulocyte interaction. Using what we believe to be optimal conditions, the effect of serum on the transferrin-reticulocyte system was re-examined. The results clearly indicated an enhancement of iron uptake by reticulocytes in the presence of serum, as well as an accelerated incorporation of iron by the cytoplasmic fraction.     

The enzymatic reduction of actinomycin D to a free radical species

Actinomycin D is an antitumor antibiotic in current clinical use. The ability of this and other antitumor antibiotics to undergo a reductive metabolism to produce free radical species has raised considerable interest in the literature in the past few years. The ability of actinomycin D to undergo a reductive metabolism was investigated using a ferredoxin reductase/NADPH system. This enzyme system has been used by a number of authors as a model for an enzymatic drug reducing system. In this study radical production was measured using direct ESR spectroscopy, the spin trapping technique, and oxygen consumption. It was shown that under anaerobic conditions the ferredoxin reductase/NADPH system could reduce actinomycin D to produce a semiquinone-imine free radical (aN = 2.8 (2N); aH = 2.8 (3H)). This radical production was found to be both drug and NADPH dependent. The effect of DNA on the drug's metabolism was also investigated. This was thought to be important because the proposed therapeutic action of the drug is centered on the DNA. Addition of calf thymus DNA to the reaction system abolished the signal produced by the actinomycin D, suggesting that intercalated actinomycin D is not a suitable substrate for ferredoxin reductase. Under aerobic conditions the ferredoxin reductase/NADPH/actinomycin D system generated the superoxide anion radical by reducing molecular oxygen. Evidence for this was obtained by spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The DMPO-superoxide radical adduct was produced (aN = 14.4 G; aH beta = 11.4 G; aH gamma = 1.3 G). Production of this adduct was drug and NADPH dependent, and was inhibited by superoxide dismutase. Superoxide production was also monitored by oxygen consumption studies.     

Comparison of the effects of zinc deprivation and actinomycin D on ribonucleic acid synthesis by stimulated lymphocytes.

1. EDTA inhibited incorporation of [3H]uridine into RNA of lymphocytes, but did not decrease uptake into the cold-acid-soluble fraction of the cells. The inhibition by EDTA was largely reversible by simultaneous addition of Zn2+. 2. Low concentrations pf actinomycin D (3 ng/ml) added at the time of stimulation of the cells inhibited [3H]uridine incorporation into RNA, but concentrations of 50-100 ng/ml were required to produce the same degree of inhibition if addition of actinomycin D was delayed until just before the incorporation was measured. This difference in sensitivity did not reg within the cells. 3. When added immediately before phytohaemagglutinin, actinomycin D (3 ng/ml) and EDTA produced similar time-courses of inhibition of uridine incorporation. 4. Uridine incorporation at 32h was inhibited when actinomycin D (3 ng/ml) or EDTA was added just before stimulation of the cells, but was only slightly affected when they were added at 32h. At intermediate times the incorporation of uridine remained sensitive to addition of EDTA for longer than it was sensitive to actinomycin D. 5. Polyacrylamide-gel separation of RNA synthesized in EDTA-treated cultures in the presence or absence of added Zn2+ showed that lower availability of Zn2+ resulted in a decreased rate of transfer of radioactivity from 32S to 28S rRNA and decreased survival of 28S rRNA relative to 18S rRNA. 6. Close similarities have been shown to exist between the effects of EDTA and low concentrations of actinomycin D. Not all the effects of EDTA could be explained by postulating that Zn2+ was a constituent of RNA polymerase I, nor were the effects of actinomycin D readily explained by previously suggested mechanisms of action of this antibiotic.     

A fluorometric method for determination of actinomycin D in serum

A sensitive fluorometric method for the determination of actinomycin D in serum has been developed. The method is based on the fluorescence of the product obtained when actinomycin D is oxidized with alkaline hydrogen peroxide. The fluorescence is measured at 420 mμ with excitation at 370 mμ. The lower limit of detection for actinomycin D is 0.1 μg of actinomycin D per milliliter of serum. In this method, actinomycin D is totally recovered from serum by extraction with ethyl acetate.     

The histology of inhibition of limb regeneration in the newt, Triturus, by actinomycin D

Adult newts, Triturus viridescens, were treated with from 1.0–10.0 μg/g body weight of actinomycin D one day before amputation of both forelimbs. Mean survival times ranged from over 50 days in newts treated with 1.0 μg/g to 13.2 days in animals given 10.0 μg/g body weight of actinomycin. Low doses little altered the course of regeneration, but animals treated with over 2.0 μg/g never formed blastemas. In another series, animals were given doses of 2.5 μg/g body weight of actinomycin D at intervals from 14 days before to 30 days after amputation. It was found that certain signs of toxicity (loss of equilibrium) are related to the time of administration of the drug whereas others (hemorrhage into the limb stumps) are restricted to a definite phase of the regenerative process. Early administration of actinomycin completely inhibits regeneration whereas later treatment results in a considerably lessened effect. The postamputational stages which are basically destructive in nature are not noticeably affected by actinomycin D, but the phases of dedifferentiation, blastema formation and redifferentiation are strongly inhibited.     

The effect of inhibition of RNA synthesis by actinomycin D on the population of basic polypeptides of the 30S unclear ribonucleoprotein particles

In rats injected intraperitoneally with actinomycin D (2 mg/kg body weight) 12 h earlier, the yield of the 30S ribonucleoprotein particles isolated from liver nuclei by extraction with 0.1 M NaCl at pH 8.0 decreased by 60 per cent. The protein-to-RNA ratio of these particles increased to 32:1 from the ratio 4.4:1 found in the same particles isolated from the nuclei of liver of control rats. The particles isolated from the liver nuclei of rats injected with actinomycin D were depleted of all charge isomers of the two most prominent polypeptides (33,000 and 39,000 daltons) present in the particles of liver of control animals. The most abundant protein in these particles was a 43,000 dalton polypeptide. This polypeptide is the least prominent of the 3 major polypeptides present in the control particles. The same charge isomers of the 43,000 dalton polypeptide were present in the nuclear ribonucleoprotein particles isolated from the liver of control animals and from the liver of animals treated with actinomycin D 12 h earlier. In control animals the nuclear ribonucleoprotein monoparticles isolated from kidney contained 3 major polypeptides of the same molecular weight with the same distribution of their charge isomers as were present in the particles isolated from liver nuclei. The injection of actinomycin D 12 h earlier was without any effect on the protein composition of the 30S nuclear ribonucleoprotein particles of rat kidney.     

Production of Actinomycin D in Complex Media

The production of actinomycin D was examined using Streptomyces antibioticus growing on a complex medium based on glucose and casein hydrolyzate. Purification of the medium to remove calcium increased actinomycin production by about one-third and prevented production of a characteristic brown pigment. The sensitivity to magnesium concentrations was less than was reported by Katz et al.,¹¹⁾ while similar requirements were found for zinc and iron.     

Some metabolic aspects of tolerance to bacterial endotoxin

Berry, L. Joe (Bryn Mawr College, Bryn Mawr, Pa.), and Dorothy S. Smythe. Some metabolic aspects of tolerance to bacterial endotoxin. J. Bacteriol. 90:970-977. 1965.-The tolerance to bacterial endotoxins which is produced in mice given a series of daily injections of heat-killed Salmonella typhimurium failed to occur when actinomycin D was administered with the heat-killed cells. Neither ethionine nor 2-thiouracil, when given with endotoxin, altered the development of tolerance. An injection of endotoxin, actinomycin D, or ethionine lowered the activity of the liver enzyme tryptophan pyrrolase more significantly at either 4 or 17 hr postinjection in normal mice than in tolerant mice. Similarly, an injection of either saccharated iron oxide or Thorotrast lowered liver tryptophan pyrrolase activity more extensively in normal than in tolerant animals. Activation of the reticuloendothelial system (RES) of tolerant mice, as determined by an accelerated rate of carbon clearance from the blood, was observed, but this was prevented by the appropriate dose of actinomycin D. Similar results were obtained when saccharated iron oxide, rather than endotoxin, was used to activate the RES, but these animals were not resistant to endotoxin and their tryptophan pyrrolase was normally diminished after an injection of endotoxin. Thus, RES activation may occur without tolerance developing. A more nearly normal level of enzyme activity appears to be characteristic of the tolerant state.     

Rapid response of protein synthesis to insulin in 3T3 cells: Effects of protein kinase C depletion and differences from the response to serum repletion

Quiescent 3T3 cells grown in media containing 4% foetal calf serum showed different responses to insulin and to serum repletion (to 12%). Insulin stimulated protein synthesis within 1 h and this early response was insensitive to actinomycin D. The later insulin response showed progressive sensitivity to actinomycin D. The serum response was slower, not occurring until 1 h, and was inhibited by actinomycin D. Depletion of cell protein kinase C by pre-treatment with phorbol ester caused a total block of the immediate response to insulin but had little effect on the response to serum or the later response to insulin. Acute phorbol ester treatment stimulated protein synthesis.     

杆状病毒凋亡抑制基因的稳定转化及抗凋亡昆虫细胞系的构建

构建了新霉素抗性基因为筛选标记的带有凋亡抑制基因p35的重组质粒p35IE1Neo, 转化Sf9细胞后, 经G418筛选得到含有p35IE1Neo的Sf9细胞, 克隆化培养后命名为Sf9-35。PCR检测表明, Sf9-35细胞的染色体DNA上有p35基因的扩增带。经放线菌素D处理后的细胞核酸电泳和TdT介导bio-DUTP缺口末端标记(TUNEL)试剂盒检测, 证实Sf9-35具有抗凋亡特性。     

Tentacle morphogenesis in hydra

Summary Hydra oligactis exposed to 3 g/ml actinomycin D for 24 hours regenerated only the first pair of tentacles (the mid-laterals). If left uncut, actinomycin D treated animals underwent a reduction of the normal number of tentacles to two or less.Inductive activity was retained in the 2-tentacled hypostomes. However, the tentacles present exhibited reduced capacities to capture and manipulate prey.Histological studies showed that the tentacles of actinomycin D treated hydra were morphologically identical to those of the controls. The interstitial cell (I-cell) population of the treated animals, however, became depleted. Replacing the hypostome of an actinomycin D treated hydra with a normal hypostome reversed the cellular effects of actinomycin D treatment.The modifications in tentacle morphogenesis occurring after actinomycin D treatment are consistent with an impairment of hypostomal function in the animal. It is suggested that the morphological site of this malfunction may be in the nervous system.Research supported by American Cancer Society Institutional Grant No. 342-9157, USPHS Institutional Grant No. 342-9241 and by a grant from Research Corporation.Part of this work was completed while L.H. was an undergraduate research student supported by the National Science Foundation.     

Studies on the Action of the Nuclear Factor Promoting Actinomycin D-Binding Capacity of Chromatin

It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.     

Studies on the action of the nuclear factor promoting actinomycin D-binding capacity of chromatin

It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.     

Stimulation of liver cholesterol synthesis by actinomycin D

1. An eightfold increase in the incorporation of [2-(14)C]acetate into liver cholesterol in vivo was observed 24hr. after starved rats had been given actinomycin D (0.5mg./kg. of body wt.). Liver cholesterol radioactivity declined faster in the treated animals, suggesting a greater rate of cholesterol turnover. 2. Liver slices from treated animals showed a tenfold increase in the incorporation of [2-(14)C]acetate into cholesterol; conversion into CO(2) and into fatty acids was less markedly increased, and conversion into ketone bodies was not significantly affected. 3. The patterns of conversion into liver cholesterol in vivo of the lactone and the sodium salt of mevalonic acid differed markedly. The former was converted at a faster rate and to a greater extent than the latter. Treatment with actinomycin D increased the conversion of both forms of mevalonic acid into liver cholesterol, but only to a small extent. 4. Stimulation of the incorporation of acetate into cholesterol occurred at 4hr. after the administration of actinomycin D but not at 2hr. The response was abolished by the simultaneous administration of dl-ethionine or puromycin. 5. Pre-feeding with a cholesterol-rich diet greatly diminished the stimulation of conversion of acetate into cholesterol caused by actinomycin D, though it did not completely suppress it. Adrenalectomized animals responded to the drug, but much less markedly. 6. It is concluded that actinomycin D stimulates the synthesis of cholesterol in the liver at a stage in the pathway before mevalonic acid, by a mechanism that probably requires protein synthesis. A likely site would be the beta-hydroxy-beta-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Some possible mechanisms by which the drug may lead to increased activity of this enzyme are considered.