Actinomycin hormonal

The induction of PAL activity at the onset of vascular differentiation can be shown by the use of plant tissue cultures (37-39). Xylem cells with secondary and lignified walls are differentiated over a time course of 3-14 days by the application of the plant growth factors naphthylene acetic acid (NAA) and kinetin in the ratio 5 1 (1.0 mg/liter NAA, 0.2 mg/liter kinetin) to tissue cultures of bean cells (Phaseolus vulgaris) (37,40). The time for differentiation varies with the type of culture, solid or suspension, and with the frequency and duration of subculture, but for any one culture it is relatively constant (37,41,42). At the time of differentiation when the xylem vessels form, the activity of PAL rises to a maximum. The rising phase of the enzyme activity was inhibited by actinomycin D and by D-2,4-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (MDMP) applied under carefully controlled conditions (42). This indicated that both transcription and translation were necessary for the response to the hormones. Experiments using an antibody for PAL and a cDNA probe for the PAL-mRNA have also shown that there is an increase in the amount of transcript for PAL during the formation of lignin when Zinnia mesophyll cells are induced to form xylem elements in culture (Lin and Northcote, unpublished work). 

Induction of de novo synthesis of a-amylase by GA in isolated aleurone layers is evident after a lag period of approximately 8 hr following administration of the hormone. In keeping with hormone responses generally, GA must be present continuously if the de novo synthesis of hydrolases is to be sustained. Synthesis of new RNA is essential to the GA-induction of de novo synthesis of hydrolases. Actinomycin D, an inhibitor of RNA synthesis, inhibits the synthesis and release of a-amylase if the inhibitor is presented during the first 7 to 8 hr after treatment. Inhibitors of protein synthesis, such as cycloheximide, also inhibit GA-induction of hydrolases. And, interestingly, abscisic acid, a growth-inhibiting hormone, inhibits GA-induced a-amylase synthesis as well. 

G20. Griffin, M. J., and Cox, R. P., Studies on the mechanism of hormonal induction of alkaline phosphatase in human cell cultures. I. Effects of Puromycin and Actinomycin D. J. Cell Biol. 29, 1-9 (1966). 

B12. Bowers, C. Y., Lee, K. L., and Schally, A. V., Effect of Actinomycin D on hormones that control the release of thyrotropin from the anterior pituitary glands of mice. Endocrinology 82, 303-310 (1968). 

Tata JR. Inhibition of the biological action of thyroid hormones by actinomycin D and puromycin. Nature 1963 197 1167-1168. 

Vitamin D functions in the process of calcium mobilization from previously formed bone making it available to the extracellular fluid upon demand by the calcium homeostatic system as described under the regulation of vitamin D metabolism. From the experiments described in the metabolism section it is clear that 1, 25-(0H)2D3, rather than 25-OH-D3 or vitamin D3 functions in the mobilization of calcium from bone under physiologic conditions. The mechanism whereby 1,25-(0H)2D3 initiates mobilization of calcium from bone is not at all understood. In contrast to the response of intestinal calcium transport to 1, 25-(0H)2D3 this process is blocked by the prior administration of actinomycin D suggesting that in fact a transcriptive event is involved in this activation As previously pointed out, bone possesses a specific receptor for 1,25bone cells has not yet been determined however. In vivo the 1,25-(OH)2D3 activation of bone calcium mobilization requires the presence of parathyroid hormone but the nature of... 

Stubbs, D. W., and D. B. Haufrect. 1968. Effects of actinomycin D and puromycin on induction of gulonolactone hydrolase by somatotrophic hormone. Arch. Biochem., 124 365. 

Estradiol administered to immature rats and rabbits preferentially stimulates the synthesis of A subunits of LDH in the uterus (Goodfriend and Kaplan, 1964). Like other effects of estradiol on uterus, this preferential stimulation of A subunit synthesis could be blocked by actinomycin D. When estradiol administration was discontinued, a return to a normal proportion of A and B subunits was observed. Despite this lack of permanence the effect of estradiol may provide a case of independent regulation of A and B subunit synthesis by differential action of the hormone on... 

The cellular target of the 1,25-hydroxylated derivative is unknown. It is still debated whether the hormone acts at the mucosal or the serosal site of the intestinal cell. A number of observations have suggested that the metabolite might stimulate calcium absorption by modifying gene expression e.g., the 7 hour time lag between metabolite administration and the increase in calcium absorption, and the intranuclear localization of the metabolite. Vitamin D is known to induce the formation of the calcium binding protein and the calcium-sensitive ATPase. However, this view must be reconciled with the fact that actinomycin D does not interfere with calcium absorption while it is known to block the conversion of the 25-hydroxylate to the 1,25-hydroxylate derivative. 

Studies on the incorporation of labeled amino acids in thyroid slices have provided a more detailed description of the mechanism of synthesis of the hormone. The radioactivity first appears in soluble polypeptides with sedimentation coefficients of 3, 8, or 12. Puromy-cin or actinomycin blocks the incorporation of the precursor into the soluble polypeptides. The half-life of the messenger RNA for thyroglobulin polypeptide was estimated to be 15-20 hours. Indeed, after inclusion of actinomycin in the incubation mixture, thyroglobulin synthesis continues for several hours. The subunits are transferred from the site of synthesis to an assembly center, in which the subunits are iodinated, carbohydrate units are included in the molecule, and subunits are condensed into a finished protein. Puromycin fails to interfere with the formation of 19 S units. 

Appealing as it is, the hypothesis that the thyroid hormone exerts its entire metabolic effect by stimulation of protein synthesis in mitochondria or in the endoplasmic recticulum is unlikely metabolic effects of the thyroid hormone continue to be expressed when actinomycin D is administered simultaneously [35]. 

Effect on Lipid Metabolism. Our knowledge of the effect of cortisone on lipid metabolism is still fragmentary. Interpretation of the results is complicated by the fact that the effect of the hormone seems to vary depending upon the source of the adipose tissue. Adrenalectomy stimulates and corticoid injections decrease lipogenesis in the adipose tissue of the mesentery. The decreased lipogenesis induced by corticosteroids is accompanied by release of free fatty acids. When corticosterone and hydrocortisone are added to epididymal adipose tissue incubated in vitro, the hormones fail to stimulate lipogenesis from [ " Cjpyruvate, but they accelerate fatty acid release, and the lipolytic effect is completetly blocked by actinomycin D. Consequently, one effect of glucocorticoids on some of the adipose tissues seems to be to accentuate lipid catabolism. 

In the acrenal, a hormone s effect on steroidogenesis concerns at least three steps in steroid metabolism (1) a protein synthesis-independent stimulation of the conversion of cholesterol ester to cholesterol (2) an actinomycin-, cycloheximide —, and puromycin-sensi-tive stimulation of the conversion of cholesterol to pregnenolone and (3) an activation of C-11 j8-hydrox-ylase, the enzyme that converts 11-deoxycorticosterone to corticosterone. 

Cycloheximide (CHI) infiltration of Cuscuta tissue was earlier shown to partially suppress the excision drop of PAL activity, suggesting a need for protein synthesis in the inactivation process [14]. The effects of CHI and actinomycin-D (Act-D) were therefore tested for their effects on both excision drop and BA + GAg-induced PAL activity increase. Table 4 shows that both inhibitors do suppress excision drop by about 85%, suggesting that de novo transcription and protein synthesis are required in the process. However, CHI or Act-D suppression of the PAL induction occasioned by use of BA + GA3 was almost complete. Thus, the increase of PAL activity on treatment with the hormones apparently involves de novo PAL synthesis. 

The effect of hormones on the A6 desaturation of linoleic, a-linolenic and oleic acids was shown as early as 1966 (Mercuri et al, 1966, 1967). It was found that alloxan diabetes depressed the A 6 desaturase activity of rat liver microsomes and insulin injection corrected the defect in less than 38 h. The simultaneous injection of actinomycin D or Puromycin that abolished protein synthesis imparted the reparative effect of insulin and suggested that insulin effect might involve A6 desaturase induction. However... 

In addition to its effects on bone and kidney cells, PTH affects the transport of calcium in the cells of the intestine and the lactating mammary gland. The hormone increases the absorption of calcium from the gut and its secretion in milk. Thus parathormone resembles vitamin D in regulating calcium transport in various kinds of cell. It too may act by regulating the biosynthesis of a specific protein, since actinomycin D prevents the action of PTH on osteoclasts which results in mobilization of bone mineral. Actinomycin D, however, does not affect the increased phosphate excretion by the kidney caused by PTH, so presumably a different mechanism is involved. 

Another level of metabolism peculiar to mammals that may be involved in mutagenesis concerns hormones. The biological action of hormones begins at the level of the chromosome. Steroids bind to a receptor in the cytoplasm of hormone-target cells the hormone-receptor complex diffuses into the nucleus and induces a modification in chromatin structure as indicated by increased nuclear binding of isotopically labeled actinomycin Thyroxine diffuses... 

There are arguments for interpreting the action of aldosterone on active sodium transport by toad bladder, colon and skin as due to a hormone-induced multiplication of the sites of penetration of sodium into the cell, across its apical border (Crabbe, 1967) this requires the intervention of nucleic acid metabolism and the effect of aldosterone is blocked by actinomycin D. This drug actually fails to interfere significantly with the reaction of toad skin to insulin before paired preparations were stimulated with insulin, short-circuit current (uk/cm S.E.) averaged 42.4 + 2.6 for the control, 36.1 1.4 for the matched fragments exposed for 2 hours to actinomycin D, 10 2 hours after addition of insulin to both... 

Protein synthesis in mitochondria is dependent on the suppty of ATR either oxidative phosphorylation, or a steady supply of ATP must be provided. From a pharmacological standpoint, it is interesting that the incorporation of amino acids is affected by th3iroid hormone in vivo. The labelled amino acids are incorporated into an insoluble protein fraction present in the membrane and none of the soluble mitochondrial enzymes studied so far become labelled to any appreciable extent. The process of protein synthesis in mitochondria, as monitored by the incorporation of amino acids, displays some peculiar characteristics it is inhibited by a variety of other amino acids, possibly due to competitive effects among different amino acids for a common transport mechanism. Also peculiar is the sensitivity to chloramphenicol, and the insensitivity to cycloheximide, which is typical of bacterial systems, and not of microsomal systems. Then, there is the observation that actinomycin-D (a known inhibitor of the nuclear DNA-dependent RNA polymerase), inhibits protein synthesis in mitochondria after treatments have been applied which affect the permeability of the membrane, thus permitting penetration of the antibiotic. This last observation indicates synthesis of messenger RNA in mitochondria via a specific DNA-dependent RNA polymerase. Protdn synthesis in mitochondria is thus apparently dependent on the continuous synthesis of RNA this is possibly due to a peculiar lability of mitochondrial messenger RNA. 

The third criterion is that suppression or inhibition of any phenomena in the cause-effect sequence of the model should produce suppression or inhibition of all the following phenomena of this sequence. In the case of ecdysone. inhibitors of nucleic acids and protein synthesis, such as actinomycin, mitomycin. F-deoxycytidine. puromycin, streptomycin, erythromycin, etc., inhibit in parallel the enhancement of DOPA decarbox ase activity, sclerotization and pupation. These results support the model, but they do not prove it Indeed, they suggest that intact nucleic acid and protein synthesis are required for the expression of the hormonal effect but they do not imply that the hormone acts through stimulation of the synthesis of some proteins and/or nucleic adds. Furthermore, inhibitors of nucleic acids and protein synthesis may have multiple sites of action, so that inhibition of a hormonal effect may be non-specific. 

Effects on protein synthesis at the translation level have also been suggested for other hormones. In the case of the action of insulin on rat diaphragm, insulin activates inert cell ribosomes. This effect requires intact protein synthesis. For other hormonal actions, the evidence in favour of control at the translation level has generally been a stimulation of protein synthesis in the presence of doses of actinomycin-D, which block the synthesis of RNA. 

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