Induction of surfactant protein in fetal lung. Effects of cAMP and dexamethasone on SAP-35 RNA and synthesis

Synthesis of surfactant-associated glycoprotein of Mr = 30,000-35,000 (SAP-35) was induced in explant culture of human fetal lung obtained from 8 to 24 weeks of gestation. SAP-35 synthesis and content increased markedly during 1-5 days in organ culture in association with the morphologic maturation of Type II epithelial cells and the appearance of lamellar bodies. [35S] Methionine labeling of the explants and subsequent immunoprecipitation of SAP-35 demonstrated distinct high-mannose precursors and sialylated SAP-35 forms as early in culture as SAP-35 synthesis was detectable. The increase in SAP-35 synthesis was associated with increased SAP-35 RNA of 2.1 kilobases as assessed by hybridization assay with [32P]cDNA specific for human SAP-35. Specific SAP-35 RNA increased during organ culture and both SAP-35 content and SAP-35 RNA increased in the absence of exogenous hormones in 2% carbon-stripped fetal calf serum. SAP-35 content and synthesis was stimulated by 8-Br-cAMP. Addition of 100 microM 8-Br-cAMP, enhanced both the concentration of SAP-35 protein and the SAP-35 RNA as assessed by hybridization assay. In contrast, treatment of the explants with dexamethasone was associated with decreased SAP-35 protein synthesis, SAP-35 content, and decreased SAP-35 RNA levels compared to untreated explants. Inhibition by dexamethasone occurred at all gestational ages tested, was dose-dependent, and detectable within 24-48 h during organ culture. Dexamethasone significantly inhibited both basal and cAMP-induced SAP-35 synthesis. Induction of pulmonary surfactant protein (SAP-35) synthesis during organ culture of human fetal lung was associated with increased SAP-35 RNA. SAP-35 synthesis and SAP-35 RNA were inhibited by dexamethasone and enhanced cAMP.     

Autoradiographic detection of HPRT variants of human lymphocytes resistant to RNA synthesis inhibition

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.     

Metabolic activity of Trypanosoma lewisi cultured in vitro in the presence of normal or ablastinic rat serum

Trypanosoma lewisi, in cultures supplemented with normal rat serum, synthesized the majority of new RNA during the first 13 hr of the culture, whereas DNA synthesis occurred from the 8th hr onwards and amino acids were incorporated into macromolecules uniformly throughout the 24 hr culture period. Thymidine was taken up by the parasite only between the 7th and 14th hr of culture, unlike uracil and amino acids which were taken up as required. Ablastin, a trypanostatic factor in the serum of infected rats, maximally inhibited DNA synthesis if it was added to the cultures before the 5th hr, partially inhibited synthesis if added between the 5th and 11th hr, but if added after this had no inhibitory effect. Ablastin only partially inhibited RNA synthesis and was without effect on amino acid utilisation.     

Effects of abscisic Acid on growth, RNA metabolism, and respiration in germinating bean axes

The effect of abscisic acid on growth, respiration, the ATP pool, and rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination has been measured in experiments where the duration of abscisic acid application and its concentration have been varied. At concentrations from 10(-7) to 10(-4)m, abscisic acid inhibits synthesis of RNA with maximal inhibition (80%) at 10(-5)m. RNA synthesis is inhibited by abscisic acid at all times examined (12, 18, and 24 hours), but the extent of inhibition is maximal at 18 hours. In 18-hour axes RNA synthesis is inhibited 42%, ATP pool size is reduced 3%, and O(2) consumption is decreased by 6% after 75 minutes of abscisic acid treatment. Inhibition of RNA synthesis is complete by 2 hours of treatment with abscisic acid, and recovery to near control levels occurs by the 3rd hour after removal from abscisic acid.     

Inhibition of RNA synthesis in embryo of Xenopus laevis by protease inhibitor

We have studied the role of proteases during the development of Xenopus laevis embryos with the aid of protease inhibitors. The activity of proteases was found to be only minimal in the unfertilized egg and during the initiation of development, but activity began to increase at the morula stage. When the activity of proteases was inhibited by antipain, an inhibitor of endopeptidase activity, RNA synthesis in the embryo was inhibited. To examine the relationship between the inhibitory effect of antipain on protease activity and its effect on RNA synthesis, antipain was reduced with NaBH4 to inactivate its protease inhibitory activity. The reduced antipain did not inhibit RNA synthesis in the embryo. Antipain effectively inhibited synthesis of both rRNA and poly(A)+RNA but not 4S RNA. We therefore suggest that protease activity plays an important role in the initiation and/or continuation of RNA synthesis.     

Neplanocin A. A cyclopentenyl analog of adenosine with specificity for inhibiting RNA methylation

The mechanism of action of the adenosine analog, neplanocin A (NPC), was investigated in human colon carcinoma cell line HT-29. Cell viability was reduced to 38 and 17% of control by 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. Cytocidal activity was not affected by inhibition of adenosine deaminase with 2'-deoxycoformycin. Concomitant with decreased cell viability was the reduced incorporation of [14C]dThd and [3H]Leu, and to a lesser extent [3H]Urd, into acid-precipitable material. Labeling of rRNA and tRNA during drug treatment for 24 h with [methyl-3H]Met and [14C]Urd revealed that NPC primarily inhibited RNA methylation, and to a lesser extent, RNA synthesis. RNase T2 digests of total RNA indicated that base and 2'-O-methylation were inhibited to approximately the same degree. Metabolites of NPC were measured by reverse-phase high-performance liquid chromatography and it was found that the major drug metabolite was the drug analog of S-adenosylmethionine with little formation of the respective, S-adenosylhomocysteine metabolite. NPC was utilized to a very small degree for RNA synthesis where only 2 and 30 pmol of NPC/A260 were incorporated into rRNA and tRNA after 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. These results indicate that NPC is metabolized to a metabolite of S-adenosylmethionine which is a poor methyl donor for RNA methyltransferases, and that the accompanying decrease in RNA methylation and protein synthesis appears to be related to its cytocidal activity.     

Reversible inhibition of ribosomal RNA synthesis in HeLa by lucanthone (Miracil D) with continued synthesis of DNA-like RNA

Ribosomal RNA synthesis was selectively inhibited in HeLa cells by lucanthone, a clinically useful schistosomicide which shares many of the properties of Actinomycin D. Synthesis of DNA-like RNA continued during complete inhibition of ribosomal RNA synthesis. Under these conditions newly synthesized DNA-like RNA accumulated normally in polyribosomes of the cell cytoplasm; most of it appeared to be messenger RNA. DNA synthesis was partially inhibited by lucanthone but protein synthesis was undisturbed. Synthesis of ribosomal RNA promptly resumed after removal of lucanthone and cell survival was not affected if exposures to the drug were limited to two hours.     

Effect of tannic acid on the synthesis of protein and nucleic acid by rat liver

1. As early as 1hr. after the intraperitoneal administration of tannic acid to rats, it could be demonstrated in the liver. At 3hr. the nuclear fraction contained the largest amount of tannic acid. 2. Nuclear RNA synthesis was inhibited in vivo 2hr. after the administration of tannic acid. Induction by cortisol of tryptophan pyrrolase was 90% inhibited at 24hr. 3. Incorporation of [1-(14)C]leucine into protein by liver slices from treated rats was decreased by 50% after 24hr. Its incorporation into postmitochondrial supernatant from treated animals was not inhibited. Incorporation into slices and postmitochondrial supernatants were inhibited in vitro by tannic acid. 4. The sequence of events: concentration of tannic acid in nuclei, inhibition of nuclear RNA synthesis, inhibition of protein synthesis and production of necrosis, is discussed.     

Sequence relationships between adenovirus 2 early RNA and viral RNA size classes synthesized at 18 hours after infection.

Synthesis of cytoplasmic viral RNA was studied during infection of cultured human (KB) cells with adenovirus 2. At 6 h, before viral DNA synthesis began 5% of the poly(A)-containing RNA hybridized to viral DNA; by 12 h and at later times more than 80% was virus specified. At 18 h after infection, four major size classes of cytoplasmic viral RNA were identified among the poly(A)-containing molecules. These size classes migrated as 27S, 24S, 19S, and 12 to 15S in polyacrylamide gels. The three larger size classes could also be identified in denaturing formamide gels. Hybridization of the 27S, 24S, and 19S viral RNAs was not inhibited by RNA harvested from cells at early times in infection. Therefore, these three major RNAs must code for late viral proteins. Hybridization of the 12 to 15S RNA was partially inhibited by RNA from cultures harvested at early times, suggesting that in this size class some of the RNA labeled at 18 h codes for early viral proteins.     

Role of RNA synthesis in DNA replication of synchronized populations of cultured mammalian cells

Using the harvesting method of synchronizing L cells, the relationship of RNA synthesis of DNA replication was studied by the use of selective inhibitors of RNA synthesis such as actinomycin D and chromomycin succinate. The synthesis of the early replicating DNA fraction is a process sensitive to the inhibition of RNA synthesis during the G1 period. The synthesis of early replicating DNA was inhibited by chromomycin succinate without affecting the initation of DNA synthesis. However, actinomycin D inhibited the synthesis of early replicating DNA and prevented the initiation of DNA synthesis in 50% of the synchronized cells. However, it was found that the continued synthesis of RNA during the S period is not essential for the synthesis of late replicating DNA. In addition to this specific response of DNA synthesis to the inhibitors of RNA synthesis, another function of early and late replicating DNA was determined relative to the cell viability. Cells synthesizing early replicating DNA were killed more efficiently by chromomycin than at other stages of the cell cycle. This indicates that the early replicating DNA unit plays a more important role in cell reproduction than the late replicating DNA unit.     

Cytotoxicity of Khat (Catha edulis) extract on cultured mammalian cells: effects on macromolecule biosynthesis

A chloroform extract of Khat (Catha edulis) leaves was used to study the cytotoxic activity on KB, 1BR.3, and XP2Bi cells. Log phase cell survival curves showed an LD50 of 40 ng/ml for KB cells. 1BR.3 and XP2Bi cells were biphasic in their response to the extract during log phase, with an LD50 of 20 and 75 ng/ml, respectively. Stationary phase cells were unaffected by the extract. DNA and RNA synthesis inhibition was studied using radiolabeled thymidine or uridine to measure the amount of extract that inhibits the synthesis to 50% of the untreated control cells. DNA synthesis was inhibited by 45, 60 and 200 ng/ml and RNA synthesis by 24, 17 and 58 ng/ml in 1BR.3, XP2Bi and KB cells, respectively. Protein synthesis was inhibited to 15-20% of untreated control cells by a dose of 40 ng/ml in all the cells studied. From this work, it is apparent that the main cause of cytotoxicity of Khat extract may be the inhibition of de novo RNA synthesis. Our results suggest that this effect is exerted on all cells used in this study and that KB cells demonstrate a higher resistance to the toxic component.     

Gibberellic Acid-enhanced synthesis and release of alpha-amylase and ribonuclease by isolated barley and aleurone layers

Gibberellic acid enhances the synthesis of α-amylase in isolated aleurone layers of barley-seeds (Hordeum vulgare var. Himalaya). In the presence of 20 mm calcium chloride the amount of enzyme obtained from isolated aleurone layers is quantitatively comparable to that of the half-seeds used in earlier studies. After a lag period of 6 to 8 hours enzyme is produced at a linear rate. Gibberellic acid does not merely trigger α-amylase synthesis, but it is continuously required during the period of enzyme formation. Enzyme synthesis is inhibited by inhibitors of protein and RNA synthesis. Small amounts of actinomycin D differentially inhibit enzyme release and enzyme synthesis suggesting 2 distinct processes. Gibberellic acid similarly enhances the formation of ribonuclease which increases linearly over a 48 hour period. During the first 24 hours the enzyme is retained by the aleurone cells and this is followed by a rapid release of ribonuclease during the next 24 hour period. The capacity to release the enzyme is generated between 20 and 28 hours after the addition of the hormone. Ribonuclease formation is inhibited by inhibitors of protein and RNA synthesis. These inhibitors also prevent the formation of the release mechanism if added at the appropriate moment.     

Several Cell Responses to Insulin of Cultured Cells Derived from Micromeres, Isolated from Sea Urchin Embryos at the 16 Cell Stage

In micromere-derived cells of sea urchin embryos, treatment with insulin started for up to 24 h during culture at 20°C resulted in augmentation of ³²P incorporation into protein (protein phosphorylation) followed by activation of ³²P incorporation into RNA (RNA synthesis) and then induced pseudopodial cable growth, accompanied by considerable decreases in the rates of protein phosphorylation and RNA synthesis. This augmentation of RNA synthesis and cable growth induced by insulin were blocked by H-7, which inhibited protein phosphorylation, and were also inhibited by actinomycin D without any inhibition of protein phosphorylation. Similar results were obtained on treatment with horse serum, found to contain insulin-like compounds. In cells treated with horse serum treated cells, high rates of protein phosphorylation and RNA synthesis were maintained even after the initiation of cable growth and about 5 h later, spicule rods were produced. Insulin treatment did not induce spicule rod formation. In cells treated with horse serum, actinomycin D treatment started at the time of initiation of cable growth, cables were formed but formation of spicule rods was blocked. These results suggest that horse serum contains some other substance besides insulin-like ones, which induces expression of genes that are indispensable for spicule rod formation.     

Inhibition of RNA- and DNA-synthesis by citrinin and its effects on DNA precursor-metabolism in V79-E cells.

1. The RNA synthesis of V79-E cells was inhibited by the mycotoxin citrinin time- and concentration-dependently. 2. Among the different RNA species mainly the rRNA synthesis was found to be inhibited by 200 microM citrinin. 3. At different precursor concentrations DNA synthesis was inhibited by citrinin after 30 min at least whereas labelling of the acid soluble fractions was found to be 3-fold higher than in untreated cells. 4. Remarkable perturbation of the DNA precursor metabolism, including release of precursor into the medium, was found to occur during citrinin treatment.     

Action of kinetin on cotyledons of fenugreek

Isolated cotyledons of fenugreek (Trigonella foenum graecum L.), which respond rapidly and specifically to the application of cytokinins with stimulated expansion, have been used to study the primary action of kinetin. Gross chemical analysis showed that ribonucleic acid increased within 24 hours in response to kinetin application. 8-Azaguanine inhibited both kinetin-induced expansion and RNA synthesis; 5-fluorodeoxyuridine inhibited only the RNA synthesis.