Biochemical paramters of BHT-induced cell growth in mouse lung.

Male mice of 7 different strains were injected i.p. with 400 mg/kg of butylated hydroxytoluene (BHT). 2 and 4 days later, the incorporation of thymidine into pulmonary DNA was significantly increased in all treated animals and this was accompanied by an increase in lung weight and pulmonary DNA. Thymidine kinase activity and DNA polymerase activity were enhanced in the lungs of BHT-treated animals and maximum activity of these enzymes appeared to precede maximum thymidine incorporation by 24 h. 3 days after BHT a good correlation was found between administered dose and thymidine kinase activity. Measuring the activity of this enzyme might serve as a convenient biochemical marker to follow and to quantitate BHT-produced cell proliferation in lung. The concentrations of cyclic AMP and the activity of adenylate cyclase were not altered by BHT on days 1-9 after administration. BHT produced also some dose-dependent, time-dependent increases in the activities of pulmonary 5'-nucleotidase and glucose-6-phosphate dehydrogenase (G6PDH), but had little effect on isocitric dehydrogenase (ICDH), pyruvate kinase (PK) and lactic dehydrogenase (LDH).     

An in vitro bioassay for a mulluscan gonadotropin.

1. Protein synthesis occurs at a high rate in the ovaries of maturing Octopus vulgaris and can be measured from the incorporation of [14C]leucine in vivo and in isolated groups of eggs in vitro. 2. Removal of the optic glands in vivo 1--3 days prior to testing markedly reduces amino acid incorporation in vivo or in vitro. After 5 days in vivo incorporation stops. 3. The rate of incorporation in vitro is increased by the addition of optic gland extract. 4. Analysis of the kinetics of leucine uptake and incorporation in vitro indicates that the hormone has an effect on the inward transport of leucine which is independent of its action on protein synthesis. 5. Electron-microscope studies of the follicle cells and ova show that the former are the site of protein synthesis. 6. Changes in either uptake or incorporation into protein by the follicle cells can be used as a qualitative biolobical assay for the optic gland hormone. Uptake is very easy to measure but incorporation is the more sensitive parameter. Either is potentially suitable as a quantitative assay for this and perhaps also for other molluscan gonadotropins.     

Effect of actinomycin D, cycloheximide, and acute blood loss on ferritin synthesis in rat liver

1. The mechanism of the stimulation of ferritin synthesis by iron in vivo has been studied in rat liver. Ferritin synthesis and turnover was measured by [(14)C]leucine incorporation. 2. Actinomycin D had no inhibitory effect, after administration of iron, on [(14)C]leucine incorporation into ferritin but appeared to augment the effect of iron on ferritin synthesis. 3. Cycloheximide completely abolished the stimulation by iron of [(14)C]leucine into ferritin and was subsequently utilized to show that iron acts in vivo by translational induction of apoferritin synthesis, rather than by stabilization of apoferritin or its precursors. 4. This conclusion was confirmed by showing that 2 days after acute bleeding, when iron was in the process of being removed from hepatic ferritin stores, ferritin synthesis was decreased whereas breakdown rates were unchanged.     

Biosynthesis and Expression of Gangliosides During Differentiation of Chick Embryo Retina Cells In Vitro

Cells from neural retina from 7-day chick embryos were cultured on polylysine-coated dishes up to 7 days. The small, round-shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GD1a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was found. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl-N-acetyl-galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.     

Heat shock modulates the ability of A-549 cell line from human lung adenocarcinoma to regulate the intensity of DNA and protein biosynthesis by an autocrine mechanism]

A-549 cells of human lung adenocarcinoma were subjected to heat shock (30 min, 44 degrees C) which caused substantial decreases in the rates of biosynthesis of the great bulk of cellular proteins with simultaneous increases in the synthesis rates of the 70 kDa protein predominantly localized in cell cytosol. By the 6th hour after the heat shock cessation this protein synthesis reached its maximum; by the 18th hour it was no longer detectable, while the protein itself was not denatured. During the recovery after the heat shock the ability of the serum-free culture medium conditioned by A-549 cells in autocrine regulation of [3H]thymidine incorporation into DNA and [3H]leucine incorporation into proteins changed also. The conditioned medium obtained within 1-3 hours after the heat shock did not influence the intensity of DNA synthesis, while the medium obtained 4-48 hours after the heat shock stimulated this process, the maximal effect (3.3-fold stimulation) being observed in the case of the 48-hour conditioned medium. Temporary (1 hour) acidification of the conditioned media down to pH 2.0 resulted in complete inhibition of the stimulating activity. Besides, these media acquired an ability to inhibit [3H]thymidine incorporation into the DNA of tracer cells. Study of effects of conditioned media on the rate of [3H]leucine incorporation into A-549 cell proteins revealed that the media obtained 1-4 hours after the heat shock inhibited this process, while the media obtained 6-18 hours thereafter stimulated it 1.2-2.1-fold. In the test systems under study temporary acidification of the media increased their stimulating influence on [3H]leucine incorporation into cellular proteins.     

Mechanisms of aneuploid induction

The effects of scavengers of active oxygen species on cadmium chloride (CdCl₂)-induced inhibition of cell growth and DNA synthesis and on the metal-induced clastogenesis were investigated to evaluate whether cadmium could induce a prooxidant state in cultured Chinese hamster V79 cells.

Inhibition by CdCl₂ of cell growth and [³H]thymidine incorporation into the acid-insoluble fraction of cells and the metal-induced clastogenesis were suppressed in part by the presence of the diffusible radical scavenger, butylated hydroxytoluene (BHT). The action of BHT was concentration-dependent and did not affect the intracellular level of cadmium. -Mannitol, a hydroxyl radical scavenger, also significantly suppressed Cd-induced inhibition of cell growth and [³H]thymidine incorporation. Catalase was marginally suppressive on Cd-induced inhibition of cell growth. These results suggest that cadmium can induce a prooxidant state in cultured mammalian cells.

The mechanism by which cadmium induces a prooxidant state was investigated by measuring the effect of cadmium on those enzymes which constitute a cellular defense against active oxygen and on the level of the intracellular antioxidant, glutathione (GSH). 2-h treatments with CdCl₂ over a concentration range of 2–10 × 10⁻⁵ M did not influence superoxide dismutase, catalase, GSH peroxidase or GSSG reductase. In contrast, the level of glutathione was decreased to approximately 40% by treatment with 2 × 10⁻⁵ M cadmium. The decrease in glutathione level may be responsible for a role by active oxygen in Cd-induced inhibition of cell growth and DNA synthesis and the metal-induced clastogenesis.     

Effect of chronic ethanol ingestion on pancreatic protein synthesis

The effect of chronic ethanol feeding on pancreatic protein synthesis was assessed by studying the rate of incorporation of [3H]leucine into proteins in isolated rat pancreatic acini in vitro. Chronic ethanol feeding increased the rate of protein synthesis (2-3-fold) compared to controls fed an isocaloric diet. The onset of the increase in protein synthesis was detectable 2 days after the beginning of ethanol feeding, reached a maximum after 7 days and remained constant for up to 4 months. The increased incorporation of [3H]leucine was not due to an increased turnover of proteins as measured in pulse-chase experiments. After separation of individual digestive enzymes by SDS-polyacrylamide gel electrophoresis and determination of the distribution of radioactivity in different proteins, a general increase in the rate of incorporation of the label into all of the proteins was observed. In contrast to the observations made with isolated acini, there was no significant difference between the control and ethanol-fed groups when the rate of pancreatic protein synthesis was measured in vivo. However, overnight withdrawal of ethanol led to an increase of approx. 70% in protein synthesis in the ethanol-fed group. These results suggest that chronic ethanol ingestion modifies the control of pancreatic protein synthesis; the enhanced protein synthesis is expressed in isolated acini, i.e., in the absence of physiological factors present during chronic ethanol ingestion and in vivo after ethanol withdrawal.     

Kinetics of insulin action on protein synthesis in isolated adipocytes. Ability of glucose to selectively desensitize the glucose transport system without altering insulin stimulation of protein synthesis

When adipocytes were exposed to [3H]leucine for times ranging from 5 to 180 s, leucine was found to enter cells rapidly and equilibrate with the cell interior within 5 s. After an additional 15-30 s [3H]leucine was incorporated into nascent protein, and the rate of incorporation was linear for up to 6 h in both control and insulin-treated cells. Since treatment of adipocytes with 10 ng/ml insulin enhanced the rate of leucine incorporation 2-3-fold with minimal or no effect on the rate of protein degradation or leucine uptake, we conclude that the predominant effect of insulin is on enhancement of protein synthesis. To assess the time required for insulin to stimulate protein synthesis, we preincubated cells with 10 ng/ml of insulin for various times from 2 to 30 min and then measured [3H]leucine incorporation into protein during a 4-min assay. These results revealed that the insulin stimulation of protein synthesis is rapid (t 1/2 of 4.4 min), but 9-fold slower than insulin activation of glucose transport (t 1/2 less than 0.5 min under identical conditions). In contrast to the rapidity of insulin activation, we found that deactivation proceeded at much slower rates (t 1/2 of 32 and 21 min for protein synthesis and glucose transport, respectively). Desensitization of the glucose transport system has previously been shown to occur after adipocytes are exposed to high glucose and insulin. To examine the specificity of desensitization, we treated cells for 6 h with 20 mM glucose and 25 ng/ml insulin and then examined insulin sensitivity and maximal insulin responsiveness of both the glucose transport and protein synthesis systems. After treatment, the glucose transport was markedly insulin-resistant (60% loss in maximal insulin responsiveness and a marked loss in insulin sensitivity), whereas the protein synthesis system exhibited neither diminished insulin responsiveness nor loss of insulin sensitivity. In fact, insulin sensitivity actually increased, as indicated by the finding that less insulin was required to stimulate protein synthesis (insulin ED50 values of 0.25 and 18 ng/ml at 0 and 6 h of treatment). From these studies we conclude that desensitization of the glucose transport system by glucose and insulin treatment appears to be specific for this particular effector system and does not reflect a state of generalized cellular insulin resistance.     

Change of isozyme pattern during activation of a transforming gene product: its relation to other biochemical markers of cellular transformation and differentiation.

Isozyme patterns of leucine aminotransferase were studied in connection with glucose transport and DNA synthesis during the activation and deactivation of the transforming gene product in rat kidney cells transformed by one Rous sarcoma virus mutant (which has a temperature-sensitive lesion in its transforming gene. On temperature shift-down of confluent transformed cells grown at 40 degrees C in the presence of fresh serum, isozyme III of leucine aminotransferase appeared in 12--20 h, with increasing amounts from 24 to 48 h. Upon temperature shift-up, isozyme I became the predominant form in these cells within 4 days, the major change occurring within the first 24 h. The rate of protein turnover was similar to the rate of loss of isozymes I and III during temperature shift-down and shift-up, respectively. A stimulation of incorporation of [3H]thymidine into DNA was observed within 8--12 h after temperature shift-down of the transformed cells. For the maintenance of stimulated DNA synthesis for at least 16 h, continued exposure to the permissive temperature is not necessary. Stimulation of glucose transport occurred prior to the stimulation of [3H]thymidine incorporation. The isozymes of leucine aminotransferase also changed during the in vitro differentiation of Yaffee L6A cells in such a way that isozyme I represented the major part of this enzyme in the fused myotube, and isozyme III was more predominant in the less differentiated state (mononucleated cells).     

VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : III. Effect of Cycloheximide on the Recovery from B12-Induced Unbalanced Growth

When cycloheximide is added to (B12)-deficient cultures before or after replenishment of the cells with B₁₂, reversion of these cells is inhibited. This inhibition is not caused by interference of the inhibitor in the uptake of B₁₂ as measured by division kinetics. Cycloheximide does not inhibit the initial increase in the rate of DNA synthesis caused by B₁₂ replenishment, but within 30–45 min the rate decreases and DNA synthesis ceases. Cycloheximide added to replenished deficient cells after completion of DNA duplication inhibits cell division. The total cellular protein and RNA in replenished cells treated with cycloheximide does not change. B₁₂ added to deficient cells does not stimulate the incorporation of [¹⁴C]leucine into protein during resumption and completion of DNA duplication. However, there is a large increase in [¹⁴C]leucine incorporation into the protein of these cells soon after completion of DNA duplication and before resumption of cell division. The addition of cycloheximide to B₁₂-replenished or to nonreplenished deficient cells rapidly inhibits the incorporation. We suggest that the addition of B₁₂ accelerates the rate of DNA synthesis in the deficient cells and that possibly no new protein synthesis is required except for mitosis. However, protein synthesis is needed for continuous DNA synthesis.     

The effects of diethylnitrosamine on ribonucleic acid and protein synthesis in the liver and lung of the Syrian golden hamster

1. Syrian golden hamsters were treated with a single subcutaneous dose of 200mg of diethylnitrosamine/kg. In the liver the treatment produced a significant and early inhibition of the incorporation of orotic acid into RNA and of leucine into protein. Diethylnitrosamine also lowered basal and 20-methylcholanthrene-stimulated activities of hepatic aryl hydrocarbon hydroxylase. 2. RNA synthesis, protein synthesis and aryl hydrocarbon hydroxylase activity were also evaluated in the lungs of the same animals. In this organ only protein synthesis was affected by diethylnitrosamine, but not RNA synthesis or aryl hydrocarbon hydroxylase activity. 3. The incorporation of thymidine into DNA was inhibited in both organs early after diethylnitrosamine treatment and increased 2–3 days later. 4. Although diethylnitrosamine, injected subcutaneously, accumulates in liver and lung in toxicologically active amounts, the acute biochemical responses of the two organs are not identical.     

除草剂普杀特（Imazethapyr）对玉米根尖蛋白质和DNA合成的影响

除草剂普杀特（Ｉｍａｚｅｔｈａｐｙｒ）对玉米根尖蛋白质和ＤＮＡ合成的影响刘支前（北京农业大学农业应用化学系,北京１０００９４）关键词：普杀特,玉米,蛋白质,ＤＮＡ８０年代美国氰胺公司开发的咪唑淋酮类除草剂,由于其高效低毒、杀草谱广,又因对某些农作物具...     

Molar proportions and relative rates of synthesis of histones in rat testis

The molar proportions and relative rates of synthesis of histones in normal and hypophysectomized rat testis seminiferous epithelial cells were determined. After hypophysectomy the molar proportions of histones H1, H2B and (H2A + protein A24) in seminiferous epithelial cells of rat testis increased while their corresponding variants TH1-x, TH2B-x and X₂ decreased, but the molar proportions of major-class histones (i.e., sum of subfractions) remained relatively constant and similar to the proportions in somatic cells. The apparent molar proportions of the labeled histones, determined immediately after 2-h periods of [³H]leucine incorporation, were much higher relative to H4 than the proportions of total histones determined by dye binding. The values, however, approached the molar proportions of total histones when rats were killed 11 days after the [³H]leucine injection. Two-dimensional gel electrophoresis confirmed that the high initial molar proportions relative to H4 by [³H]leucine incorporation were not due to the possible contamination by highly-labeled non-histone proteins. The specific activity of histone H4 relative to the specific activity of DNA, determined immediately after 3-h periods of [³H]leucine and [¹⁴C]thymidine incorporations was similar to the value when rats were killed 13 days after the injections. It is proposed that histones of seminiferous epithelial cells are synthesized disproportionally relative to H4 and in excess of the quantities required for polynucleosome assembly. The excess histones are subsequently displaced or degraded slowly.     

Lethla effect of nitrous acid on Escherichia coli.

The effect of nitrous acid (NA) on viability, integrity of cellular DNA and on membrane transport were studied in 5 strains of Escherichia coli. Stationary phase cells, grown on mineral salts medium, were exposed to NA. The viability of strains decreased in thefollowing order: W3110 wild-type greater than WP2 wild-type, WP2 uvrA greater than NG30 recA greater than P3478 polA. Alterations were found in the DNA sedimentation profile in alkaline sucrose gradient. Disturbance of DNA synthesis was measured by 3H-labelled thymidine ([3H]Thd) incorporation. No degradation of DNA was found after NA treatment. Low doses of NA caused significant inhibition of leucine and glucose transport into whole cells. The results are interpreted in terms of the multi-target action of NA causing the death of cells.     

Effect of cobalt on the synthesis and degradation of hepatic catalse in vivo

1. The administration of CoCl(2) to rats caused a decrease in hepatic catalase activity as well as a decrease in the amount of catalase protein as measured by immunological assay. The mitochondrial enzyme decreased progressively over 2 days, whereas the cytosol enzyme decreased over 12h and then remained essentially unchanged for 2 days after a single injection of cobalt. 2. Incorporation of [(14)C]glycine into catalase haem was dramatically decreased by a single injection of cobalt, but that into catalase protein remained essentially unaltered. 3. Incorporation of [(3)H]leucine into liver protein increased in rats in a steady state receiving a daily injection of cobalt, which was in contrast with a marked inhibition observed in 5-amino[(3)H]laevulinate incorporation. 4. The initial rate of [(3)H]leucine incorporation into mitochondrial and cytosol catalase did not alter or was slightly depressed in the cobalt-treated animals, whereas the incorporation of 5-amino[(3)H]laevulinate into mitochondrial and cytosol catalase was conspicuously decreased, indicating that haem synthesis was limiting catalase formation. 5. The degradation rate of catalase protein, as measured by a double-labelling method, was not changed by the cobalt treatment.     

Mitochondrial protein synthesis in chinese hamster cells (line CHO) synchronized by isoleucine deprivation

Mitochondrial protein synthesis was measured in line CHO cells after phases of the cell cycle were synchronized by isoleucine deprivation or mitotic selection. Maximum incorporation of [³H] leucine into mitochondrial polypeptides occurred within 2 hours after isoleucine was added to initiate G₁ traverse. In cells synchronized in G₁ by mitotic selection, the rate of mitochondrial protein synthesis was fairly constant throughout the cell cycle. SDS-polyacrylamide gel electrophoretic profiles of labeled mitochondrial polypeptides were similar in cells synchronized by either isoleucine deprivation or mitotic selection. Obvious changes in the distribution of polypeptides were not detected during various phases of the cell cycle. The increased rate of incorporation of [³H] leucine into mitochondrial polypeptides after reversal of G₁-arrest may indicate that mitochondrial protein synthesis and possibly mitochondrial biogenesis are synchronized in CHO cells deprived of isoleucine.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

Giant cell formation in ectopic mouse trophoblast

Segmenting mouse ova, grafted beneath the kidney capsule of syngenic adult recipients, result in a growth of trophoblast, which changes from small, actively-dividing cells into giant trophoblast cells which degenerate 15 days after grafting. Similar giant cells are found in normal mouse placentas. Radioautography with ³H-thymidine, uridine, and leucine revealed cessation of DNA synthesis after day 8, with decline in RNA synthesis from day 10, and continued protein synthesis through day 15. Treatment with Colcemid reduced the graft size but failed to suppress giant cell formation. Treatment on days 4–7 of grafting with 5-fluorodeoxyuridine (FUdR), cyclohexamide, or actinomycin D resulted in giant cell suppression with the maintenance of healthy-appearing small trophoblast cells. These results confirm the early withdrawal of trophoblast grafts from the mitotic pool and the non-mitotic increase of trophoblast DNA, and demonstrate the apparent need for RNA and protein synthesis to support the development of trophoblast giant cells.