Application Of Actinomycin Research Articles

GSK3ß, a centre-staged kinase in neuropsychiatric disorders, modulates long term memory by inhibitory phosphorylation at Serine-9

Accumulating evidence implicates deregulation of GSK3ß as a converging pathological event in Alzheimer's disease and in neuropsychiatric disorders, including bipolar disorder and schizophrenia. Although these neurological disorders share cognitive dysfunction as a hallmark, the role of GSK3ß in learning and memory remains to be explored in depth. We here report increased phosphorylation of GSK3ß at Serine-9 following cognitive training in two different hippocampus dependent cognitive tasks, i.e. inhibitory avoidance and novel object recognition task. Conversely, transgenic mice expressing the phosphorylation defective mutant GSK3ß[S9A] show impaired memory in these tasks. Furthermore, GSK3ß[S9A] mice displayed impaired hippocampal L-LTP and facilitated LTD. Application of actinomycin, but not anisomycin, mimicked GSK3ß[S9A] induced defects in L-LTP, suggesting that transcriptional activation is affected. This was further supported by decreased expression of the immediate early gene c-Fos, a target gene of CREB. The combined data demonstrate a role for GSK3ß in long term memory formation, by inhibitory phosphorylation at Serine-9. The findings are fundamentally important and relevant in the search for therapeutic strategies in neurological disorders associated with cognitive impairment and deregulated GSK3ß signaling, including AD, bipolar disorder and schizophrenia.

Long-term synaptic facilitation in defensive behavior command neurons in the snail during acquisition of sensitization depends on RNA synthesis

Experiments on defensive behavior command neuron LP11 in semi-intact common snails preparations were performed to study transcription processes underlying the mechanisms of neuronal plasticity during acquisition of nociceptive sensitization. Application of nociceptive stimuli to the heads of control snails led to increases in neuron membrane excitability, along with biphasic changes in responses to tactile and chemical sensory stimulation--with depressed responses at the short-term stage and facilitation in the long-term stage of sensitization. Acquisition of sensitization after application of the RNA synthesis inhibitor actinomycin D (20 microM) to neuron LP11 led to complete suppression of synaptic facilitation in responses to sensory stimulation in the long-term stage of sensitization, but had no effect on the short-term depression of responses. However, the inhibitor had no effect on changes in membrane excitability in these conditions. Application of actinomycin D 30 min after the end of the sensitization acquisition process (1 h after the process started) had no effect on membrane excitability or synaptic facilitation. It is suggested that the induction of long-term synaptic facilitation in neuron LP11 during acquisition of nociceptive sensitization is dependent on RNA synthesis occurring in a narrow time interval, while changes in membrane excitability during the 2-3 h after acquisition of sensitization and short-term synaptic plasticity are independent of transcription processes.

Transcriptional and Posttranscriptional Regulation of Dormancy-Associated Gene Expression by Afterripening in Wild Oat

To investigate whether the afterripening-induced changes in gene expression are at the transcriptional or posttranscriptional level in wild oat (Avena fatua) seeds, we chose four dormancy-associated genes to estimate their relative transcription activities and the stability of their corresponding transcripts in afterripened and dormant embryos. The transcription activities for those genes were 1.5 to 7 times higher in dormant embryos than in afterripened embryos 24 h after incubation, as determined by nuclear run-on assays. The half-lives of the transcripts in afterripened and dormant embryos were estimated by the use of actinomycin D. The application of actinomycin D resulted in the stabilization of the transcripts. Nevertheless, the results indicated that the half-lives of the transcripts were much greater in dormant embryos than in afterripened embryos. Considering the great differences in the steady-state levels and the half-lives of the mRNAs, and the relatively small differences in transcription activities of the genes between afterripened and dormant embryos, we conclude that afterripening regulates the expression of dormancy-associated genes in excised embryos mainly at the posttranscriptional level and that transcriptional control plays a minor role.

Haemolymph immune proteins protect the insect body cavity from invading bacteria

Injections of living, non-pathogenic Enterobacter cloacae into the body cavity of Galleria mellonella provoked the insect's non-self response system to produce antibacterial activity that eliminated the bacterial infection. Immunized larvae of Galleria contained no E. cloacae in haemolymph within 24 hr post-infection and they transmitted normally to pupae. Parallel to this protective immunity, antibacterial activity of cecropin-like polypeptides and manifold increased innate titer of haemolymph lysozyme were shown in vaccinated insects. Application of actinomycin D or cycloheximide in an induction phase of antibacterial response at a dose of 0.25 × ld 50 demonstrably reduced the lysozyme titer and entirely prevented (except for actinomycin-treated pupae) the synthesis of cecropin-like antibacterial activity. In insects with impaired antibacterial immunity, the immunizing bacteria multiplied to the titer of almost 10 9 cells/ml of haemolymph, causing death in nearly 100% of larvae due to E. cloacae bacteriaemia. No bacteriaemia of E. cloacae could be induced in larvae given actinomycin D (or cycloheximide) in an effector phase of immune response when the antibacterial proteins begin to be synthesized in invaded insects. For the expression of cell-free immunity in Galleria, there is a requirement for the de novo synthesis of antibacterial immune proteins that normally protect the insect body cavity from invading bacteria. The anti-cecropin agent from Heterorhabditis bacteriophora selectively destroyed the inducible antibacterial immunity, thereby allowing the immunizing bacteria to grow in Galleria. This implies that the cecropin antibacterial activity is primarily responsible for the elimination of E. cloacae and other bacterial invaders from the insect body cavity.

Synthesis and processing of ribosomal RNA in the Growing oocytes of Calliphora erythrocephala

The processing of the high molecular weight primary transcript and intermediates of rRNA have been analyzed after labelling in vivo the oocytes of the blowfly Calliphora erythrocephala which develop in a strictly synchronous manner. The primary 35S transcript ( 2.8 × 10 6 dalton) can be identified after less than 5 min of labelling and is subsequently cleaved, perhaps via a slightly smaller and very short lived 34S intermediate, with a half life time of less than 4 min, to yield the mature 19S rRNA ( 0.7 × 10 6 dalton) and a 29S ( 1.8 × 10 6 dalton) intermediate precursor of the 26S rRNA. The production of the final 26S rRNA, (26S m), with a molecular weight of 1.44 × 10 6 is mediated by a precursor of 1.49 × 10 6 dalton (27S) with a half life time of some 28 min. Mature 26S rRNA differs from this molecule by a further size reduction of 5 × 10 4 dalton and by an intramolecular cut which generates two hydrogen-bonded submolecules of 26S m rRNA with estimated molecular weights of 0.6 × 10 6 and 0.78 × 10 6 . They can be separated from one another and from 19S rRNA by polyacrylamide gel electrophoreses after denaturation of the 26S m rRNA. Besides the “internal break” two further cleavages occur to produce the small 5.8S and 2S submolecules of 26S m rRNA. These moieties can also be released by thermal denaturation or treatment of 26S m rRNA with hydrogen-bond interfering agencies. The order of appearance of the primary transcript and intermediates after pulse labelling and the shift of labelling towards fractions of lower molecular weights after application of actinomycin D suggests a scheme for the in vivo processing during the maturation of ribosomal RNA in C. erythrocephala.

Fast ephemeral DNA damage upon BβP injection

Injection of high concentrations of benzo[α]pyrene (10–125 μg BαP/g body weight) into young frogs ( Xenopus laevis), fish ( Gambusia affinis) or tissue culture cells (L 5178 Y) in a state of logarithmic growth causes alterations in DNA. In frogs these alterations reach a maximum at 60–90 min after application, then they decrease and become undetectable after 3 h. Within 8–12 days after the single BαP dose, a new wave of DNA alterations can again be detected. Parallel measurements of mixed function oxygenases (MFO) showed no short term activity changes within the first day. There was, however, an increase of activity starting on the 8th day following injection in frogs, and lasting until day 14. DNA alterations have been assessed by two methods. One is a modification of the alkaline filter elution method of Kohn et al. (1976) and is believed to determine the number of events leading to single strand breaks at alkaline pH. The other method determines the number of S 1 nuclease sensitive sites in highly purified native DNA. Both results were found to be highly correlated. The effect does not seem to be caused by an impurity, unless an unknown, very minor, and extraordinarily active component is present. The effect in frogs is linked to nutritional status. Frogs fed a carbohydrate-rich, protein-poor diet do not give the fast response. The fast effect cannot be blocked by application of Actinomycin, Cordycepin, and Chloramphicol injected either 30 min before or simultaneously with the B aP. Benzo( e)pyrene, a non-tumorigenic isomer of B aP, does not cause any effects at similar doses.

Characteristics and Mode of Action of a Virus Inhibitor from Cuscuta reflexa Plants

Summary The aqueous extract from the filaments of Cuscuta reflexa has shown a broad spectrum and highly significant antiviral activity. Treatment of the inhibitor on the two basal leaves of several hypersentitive hosts, prior to inoculation of viruses (isometric and anisometric), resulted in gradual increase in virus inhibition with the increase in time interval between treatment and virus challenge up to 24 hrs., thereafter the resistance of the host plants gradually decreased. In post inoculation treatments, it was effective only when applied up to 2 hrs. following virus inoculation. Along with the high degree of local inhibitory response, systemic acquired resistance (resistance at non-treated sites) was also observed in different host—virus combinations. The resistance induced (both localized and systemic) by C. reflexa inhibitor was significantly reversed by the application of actinomycin D up to 6 hrs. after treatment. AD had no effect when applied 24 hrs. after treatment with the inhibitor. The maximum inhibition was achieved by the inhibitor from the filaments of mature plants of C. reflexa, parasitizing on Zizyphus jujuba. The resistance induced by C. reflexa inhibitor was sensitive to high temperature. Extract from C. reflexa filaments was inhibitory up to a dilution of 1: 100, inactivated at 70 °C, and survived at room temperature for 12 days. The inhibitory material in crude extract was non-dialyzable, not sedimented at 120,000 g for 120 min, precipitated by a saturated solution of ammonium sulphate or ethanol, inactivated by trichloroacetic acid, adsorbed on animal charcoal, wood charcoal or celite, active at pH 10, and insoluble in diethyl ether, petroleum ether, chloroform or benzene.

Induction by hormones of phenylalanine ammonia-lyase in bean-cell suspension cultures. Inhibition of superinduction by actinomycin D.

Transfer of dark‐grown suspension cultures of Phaseolus vulgaris cells from a maintenance medium to medium with reduced auxin and increased cytokinin content resulted in an increase in phenylalanine ammonialyase activity 3–5 days later. This increase in phenylalanine ammonia‐lyase activity may be used to examine changes in control of gene expression following plant hormone action.An inhibitor of transcription (aetinomycin D) and an inhibitor of translation [d‐2‐(4‐methyl‐2,6‐dinitroanilino)‐N‐methylpropionamide) were used to investigate this phenylalanine ammonia‐lyase response under conditions of controlled cell density and optimised inhibitor dose.The rising phase of the enzyme activity was inhibited by either actinomycin D or the propionamide derivative while cytoplasmic streaming was unaffected by these inhibitors. This indicates that both transcription and translation are required for the response.The falling phase of phenylalanine ammonia‐lyase specific activity is due to inactivation or degradation of the enzyme since the decrease cannot be explained by dilution out of the enzyme during cell growth. This falling phase was insensitive to the propionamide inhibitor, but application of actinomycin D resulted in an increase in phenylalanine ammonia‐lyase activity (superinduction) after 12 h.Superinduction by actinomycin D suggests that availability of phenylalanine ammonia‐lyase mRNA for translation may be controlled to produce the change in enzyme activity. Lack of superinduction by the propionamide inhibitor indicates that translation of a protein which degrades either phenylalanine ammonia‐lyase or its mRNA is not required to cause the fall in phenylalanine ammonia‐lyase activity. The superinduction is, therefore, probably caused by an alteration in competition between mRNAs and the increased enzyme synthesis results from preferential translation of relatively stable phenylalanine ammonia‐lyase mRNA already transcribed during the rising phase of activity. Actinomycin D is postulated to suppress competition from other less stable mRNAs for the translation machinery, by inhibiting their transcription.A model based on de novo synthesis of both phenylalanine ammonia‐lyase mRNA and its encoded enzyme has been shown to apply to other phenylalanine ammonia‐lyase responses. This model assumes a constant rate of degradation of the enzyme throughout the response, and is only compatible with the mRNA‐competition hypothesis for superinduction.

Antiviral activity of Boerhaavia diffusa root extract and the physical properties of the virus inhibitor

The aqueous extract of air-dried roots of Boerhaavia diffusa shows broad-spectrum antiviral activity and no phytoxic effects. Infection by four viruses was completely prevented, at treated and nontreated sites, when the extract was applied on two basal leaves of host plants 24 h prior to virus inoculation. This inhibition was completely reversed by the application of actinomycin D on treated leaves within 6 h of extract treatment and partially reversed within 18 h. The crude extract from resistant leaves contained an inhibitor of virus infection.The inhibitor in the root extract was partially active up to a dilution of 1:500, was completely inactivated at 95 °C for 10 min, and survived at room temperature for 20 days. The expression of inhibitory activity was prevented when the treated plants were exposed to temperatures beyond 35 °C. The inhibitory principle in the extract was nondialyzable and insoluble in organic solvents, viz., petroleum ether, solvent ether, chloroform, and benzene. It was adsorbed by animal charcoal, wood charcoal, and celite, and was precipitated by ammonium sulphate (90%), ethanol (50%), and cold trichloroacetic acid (10%). The inhibitor was not sedimented at 120 000 × g for 120 min. Further characterization is being done for positive identification of the inhibitor.

Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics.

Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.

Study of nucleolar vacuolation and RNA synthesis in embryonic root cells of Zea mays.

ABSTRACT The process of nucleolar vacuolation has been quantitatively studied in root cells of the embryo of Zea mays. During germination, we have estimated the percentage of vacuolated nucleoli, the number of vacuoles per nucleolus and the volume density of the vacuoles, the latter by application of the stereological principles of morphometry. In the dormant embryo, no vacuoles can be detected in the nucleolus. When germination occurs at 22 or 16 °C, there is a rapid increase in the percentage of vacuolated nucleoli and in the volume density (VA), with a maximum after 4 and 8 h. At 6 °C, a temperature which does not permit emergence of the root, the percentage of vacuolated nucleoli increases regularly but never reaches the level observed at 16 and 22 °C. In parallel with the appearance of nucleolar vacuoles, the size of the nucleolus decreases statistically and shows a minimum when vacuolation is maximum. This suggests that the appearance of vacuoles within the nucleolus is the result of loss of material from this organelle. Indeed, electron micrographs show that granular components are lost from the nucleolus during the first 8 h of germination at 16 °C. According to the literature, these granular components are probably ribosomal subunits. An autoradiographic study of tritiated uridine incorporation shows that the nucleolus does not synthesize rRNA at the early beginning of germination. Hence the nucleolus is unable for several hours to rebuild the lost granular elements. Application of actinomycin D, 5-fluoro-uracil and 2-thio-uracil to the seeds, during the first 48 h of soaking, inhibits rRNA synthesis, the vacuolation process, the loss of granular components and the reduction in size of the nucleolus. These results allow us to determine some of the physiological conditions characterizing the appearance of the nucleolar vacuoles.

Inhibitor-dependent, reciprocal changes in the activities of glyceraldehyde 3-phosphate dehydrogenases in Sinapis alba cotyledons

Summary Glyceraldehyde 3-phosphate dehydrogenases (GPD) from cotyledons of far-red- light-grown Sinapis alba seedlings can be separated by ammonium sulfate chromatography into two enzymes designated NAD-GPD I and NADP-GPD • NAD-GPD II. The former species is probably the NAD-GPD of cytoplasmic glycolysis (EC 1.2.1.12). The latter species has both NADP and NAD activity and is apparently identical to the NADP-GPD of plastids (EC 1.2.1.13). When puromycin (100 μg • ml-1) or D-threo chloramphenicol (200 μg • ml-1) is applied to seedlings, the light-dependent increase in the activities of the bifunctional enzyme is strongly inhibited. The resultant decrease in NAD-GPD II activity of this enzyme is accompanied by an equivalent increase in NAD-GPD I activity. The application of actinomycin D (10 μg • ml-1) affects neither of these two enzymes, but significantly inhibits the activity increase of the control enzyme glyoxylic acid reductase (EC 1.1.1.26). These and other regulatory data published previously are discussed on the basis of a model proposing that the formation of glyceraldehyde 3-phosphate dehydrogenases proceeds sequentially via an anabolic pathway in which the glycolytic enzyme of the cytoplasm functions as a precursor or subunit of the bifunctional chloroplast enzyme.

Studies on Phytoalexins

Actinomycin D stimulated phaseollin production in endocarp tissues of the French bean (Phaseolus vulgaris L.), maximum production being obtained with 25 to 30 micrograms per milliliter of antibiotic. Under these conditions, net incorporation of (3)H-uridine into total cell ribonucleic acid was inhibited by more than 80% over a 6-hour induction period. If allowance was made for a 2-hour lag in the action of actinomycin D, inhibition of incorporation was greater than 95%. Contrary to other reports, no evidence was obtained of an increased formation of any specific ribonucleic acid fraction. Actinomycin D applied in the cold (4 C) was not found to be effective in stimulating phaseollin production. When applied in this way, actinomycin D did not affect induction of phaseollin by a fungal peptide, Monilicolin A, although ribonucleic acid synthesis was inhibited by more than 95%. It is suggested that the induced formation of phytoalexins may not be dependent on increased ribonucleic acid synthesis as has previously been claimed.Other experiments indicated that the apparent effects of actinomycin D on ribonucleic acid synthesis could be influenced by the choice of precursor used to label ribonucleic acid and by the order of addition of precursor and antibiotic to the plant tissue. These effects were only observed in the period immediately following application of actinomycin D. It is suggested that such effects could critically influence the results obtained in short term experiments and may explain some differences in reported action of actinomycin D from different laboratories.

Juvenile hormone-controlled synthesis of female-specific protein in the cockroach Leucophaea maderae

Application of 1 μg of the all-trans methyl (±)-12,14-dihomojuvenate induces the de novo synthesis of a female-specific protein at a rate similar to that found in unoperated egg-maturing females. JH also appears to stimulate an increased synthesis of the non-sex-specific proteins. The female-specific protein is essential for egg maturation since it makes up 85–90% of the egg proteins. The exact role of the non-sex-specific proteins in egg maturation processes is not known. 12,14-Dihomojuvenate and 12-homojuvenate, the two naturally occurring JHs, have similar potencies in inducing the synthesis of female-specific protein. The 14-homojuvenate, not known to occur naturally, also has similar potency. Epoxyfarnesenic acid is 10–20 times less potent. The all-trans isomers of either of the two JHs appear to be 2–10 times more potent than the naturally occurring hormones, whereas the t,c,t-isomer of the 12,14-dihomojuvenate has no activity. The JHs possibly influence transcription of specific mRNA since application of actinomycin D (2 μg) simultaneously with JH completely blocked female-specific protein synthesis even when assayed 5 days later. Messenger for the specific female protein is relatively long lived (at least 3–4 days).

Alterations of enzymatic activities during muscle differentiation in vitro

The activity of creatine phosphokinase, myokinase, and glycogen phosphorylase in muscle cells grown under controlled culture conditions was investigated. Cell fusion and formation of multinucleated muscle fibers were found to be closely associated with a severalfold increase in enzyme activity. The correlation is manifested both in primary cultures and during the differentiation of myogenic cell lines which were maintained for several months under conditions of continuous multiplication. Cytochemical staining for glycogen phosphorylase showed that the increased activity is localized within the multinucleated fibers. Experiments in which the duration of cell fusion was controlled by the concentration of Ca 2+ in the nutritional medium suggested that the increase in enzyme activity is largely dependent on the continuation of the fusion process. Application of actinomycin D to cultures during the stage of rapid increase in enzymatic activity did not prevent activity from increasing for several hours, whereas application of inhibitors of protein synthesis did inhibit the increase in activity. In the presence of cyclohexamide, the process of cell fusion was almost totally inhibited while in the presence of actinomycin D fusion continued for several hours. These experiments suggest the possibility that the messenger RNA molecules which specify the synthesis of proteins essential for cell fusion and increased enzymatic activity are formed at a developmental stage preceding cell fusion.

Specificity of Deoxyribonucleic Acid Intercalating Compounds in the Control of Phenylalanine Ammonia Lyase and Pisatin Levels

Compounds with planar triple ring systems such as acridine orange, 9-amino acridine, 9-amino-1,2,3,4-tetrahydroacridine (tacrine), 6,9-diamino-2-ethoxyacridine lactate monohydrate (DE-acridine), 6-chloro-9-(3'-diethylamino-2'-hydroxypropylamino) -2-methoxyacridine.2 HCl (CDM-acridine), quinacrine, 6-chloro-9-(4'-diethylamino-1'-methylbutylamino) -2-methoxy-1,10-diazaanthracene (CDM 1,10-diazaanthracene), thionine, azure A, methylene blue, and pyronine Y when applied to excised pea pods were potent inducers of phenylalanine ammonia lyase or of pisatin, or of both. Compounds with an array of structural variation around the planar three-ring system were tested for their ability to induce these responses in pea tissue. In general, dimethylamino, diethylamino, or amino substitutions at position 2 and 6 or an amino (with or without an aliphatic side chain) substitution at position 9 of the three-ring system augmented induction potential. Methyl green, methylene blue, 2,7-diaminofluorene, nile blue, neutral red, pyrogallol red, ethidium bromide, nogalamycin, quinine, chloroquine, spermine, 8-azaguanine, gliotoxin, chromomycin A(3), actinomycin D, and mitomycin C were also potent inducers. The inhibition of phenylalanine ammonia lyase induction by the application of actinomycin D (300 micrograms per milliliter) or 6-methylpurine (1 milligram per milliliter) within 1 hour after inducer application indicated that newly synthesized RNA is necessary for induction. Phenylalanine ammonia lyase induction was also inhibited by cycloheximide (150 micrograms per milliliter).

Modification of regeneration in Dugesia tigrina by actinomycin D.

AbstractDifferences in regeneration of isolated postpharyngeal body sections were observed in two laboratory groups of Dugesia tigrina. One group regenerated 14.8% of bipharyngeal specimens, which in some cases, also showed abnormal polarity. The other group regenerated only 1.19% of bipharyngeal forms under similar conditions, with no indications of seriously deviant polarity.In the first strain, the application of actinomycin D at early stages of regeneration of the body sections increased greatly the incidence of multipharyngeal regenerates (with 2–5 pharynges); it also slightly enhanced the occurrence of abnormal polarity in the reconstituted worms. In the second strain, the increase in percentage of bipharyngeal forms regenerated from similar body fragments was insignificant (up to 5%). It was without effect on either strain if introduced to the regenerating sections at later stages of regeneraton. A test for incorporation of 32P into RNA showed that actinomycin, as used in the present study, suppressed over 90% of synthesis of total RNA in the regenerating body fragments.From these findings a hypothetical model is developed to explain reorganization in the isolated postpharyngeal section as a sequence of certain local inductions and inhibitions of differentiation. The significance of synthesis of RNA in relation to these developmental events is discussed.

The influence of chlorpromazine and octoclothepine on the incorporation of 14C-glycine into brain and liver proteins in mice

A single intravenous injection of chlorpromazine or octoclothepine (10 mg/kg) enhanced the in vivo incorporation of labeled glycine into brain and liver proteins. The stimulation may be blocked by the application of actinomycin C or by adrenalectomy. Chlorpromazine and octoclothepine at a concentration of 10 −3 M inhibited protein synthesis of brain and liver slices by approximately 50%. Brain cells accumulated glycine against a concentration gradient. The active transport was inhibited by both drugs at 10 −4 − 10 −3 M. Liver cells do not accumulate glycine and the passage into the cells was not affected by the drugs.

Uterine alkaline phosphatase: effect of actinomycin D and histones.

The effects of intrauterine application of actinomycin D and calf thymus histones on the akaline phosphatase activity and histological changes of the uteri of castrated rats stimulated with 17β-estradiol were studied. The uterine phosphatase activity induced with 17β-estradiol was suppressed nearly to the level of castrated rats at high doses of actinomycin D (6 μg/horn). Although the epithelial cells were completely denuded, histochemical localization of phosphatase activity was demonstrated in the blood vessels, glandular epithelium and stroma. Lower doses of actinomycin D inhibited the height of the epithelial cells induced by 17β- estradiol and an unexplained rise in the phosphatase activity above the control level was observed. Calf thymus histones administered into the uterine cavity incited an acute inflammatory reaction and did not inhibit the increase in uterine alkaline phosphatase induced by 17β- estradiol. (Endocrinology 82: 1069, 1968)

Autoradiographic study of the incorporation of uridine- 3H during myogenesis in tissue culture

Autoradiographic investigation of differentiating thigh muscle tissue cultures showed that after brief exposures to radioactive uridine or cytosine, the amount of labeling of nuclei within muscle fibers was significantly lower than the labeling of nuclei within the mononucleated cells. The decrease in the rate of labeling which occurred at the onset of fusion of the myoblast into multinucleated fibers cannot be ascribed to greater absorption of the β rays by the cytoplasm of differentiated cells nor is it due to lack of penetration of the labeled precursor into the cells. The difference in the incorporation of RNA precursors also does not seem to arise from differences in the mitotic activity of the various cell types, since inhibition of mitosis by X-irradiation did not abolish the differential rate of incorporation of uridine into RNA. The incorporation of 3H-leucine, unlike that of radioactive uridine, does not seem to decrease following cell fusion. Application of actinomycin D resulted in a higher inhibition of amino acid incorporation into mononucleated cells compared with that into multinucleated fibers. The significance of these results in relation to the possible formation of a stable type of RNA during differentiation is discussed.