Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

The molecular toxicity of monotertiarybutylhydroquionone (TBHQ) was studied using $\text{Tetrahymena}$$\text{pyriformis}$ as a model cell system. TBHQ at 26 ppm in the media inhibited cell growth by 50%. TBHQ inhibited the oxidation of ¹⁴C-acetate to ¹⁴CO₂. In addition, increasing concentrations of TBHQ decreased the incorporation of ¹⁴C-acetate into lipids and protein, ¹⁴C-amino acids into protein, ³H-uridine into RNA and ³H-thymidine into DNA. The incorporation of ¹⁴C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ.     

Some ultrastructural effects of testosterone and insulin on the ventral prostate of rats in organ culture

Summary The fine structure of the secretory epithelial cells of rat's ventral prostate has been studied following organ culture. Culturing with either testosterone or insulin alone, and with the two hormones combined, were carried out to investigate how insulin modifies the action of testosterone on the maintenance of cellular integrity. After 4 days in hormone-free culture, the secretory epithelial cells showed signs of cellular atrophy and regression, involving loss of the apical microvilli, absence of the apical secretory vacuoles, atrophy of the Golgi apparatus, decrease in rough endoplasmic reticulum and the appearance of autophagic vacuoles. The presence in the medium of either testosterone or insulin alone, or combined, prevented cellular atrophy and regression. The best maintenance of cellular integrity was obtained in a culture containing both hormones. The effects of insulin was approximately equivalent to those of testosterone in the maintenance of cellular integrity.     

Metabolism ofAedes aegypti cells grown in vitro. 1. Incorporation of3H-uridine and14C-leucine

Summary Metabolic activity ofA. aegypti cells grown in vitro has been studied by incorporation of³H-uridine and¹⁴C-leucine. “Chase” experiments with unlabeled precursors, and the use of actinomycin D and puromycin, showed that³H-uridine was incorporated into cellular RNA, and that¹⁴C-leucine was incorporated into protein of these cells. Incorporation of³H-uridine was inhibited when actinomycin D was used at a concentration of 10 μg/ml, and¹⁴C-leucine incorporation was inhibited to the same extent by puromycin at a concentration of 100 μg/ml medium. Contribution No. 148.     

Intervarietal differences in some metabolic functions associated with protein accumulation in rice grains

Summary Total nitrogen, amino nitrogen, glutamic acid dehydrogenase (GDH) activity and incorporation of ³H-uridine and ¹⁴C-amino acids into RNA and proteins, respectively, were compared in the developing grains of three high-protein stocks (IR-480-5-9, GMPR-51 and Erythroceros) and a high-yielding, medium-protein cultivar IR-8. The above parameters were also independently studied in the developing grains of IR-8 grown at 0, 60 and 120 kgN/ha. In addition, mobilization of nitrogen from flag leaf during kernel development was compared in a separate experiment. Higher protein concentration, both in high-protein stocks and in IR-8 grown at 120 kgN/ha, was associated with increased levels of: soluble amino nitrogen, GDH activity, ³H-uridine and ¹⁴C-amino acid incorporation. Significant variation was found among the high protein stocks in mobilization of nitrogen from flag leaf.Research partly supported by International Atomic Energy Agency Research Contract 1035     

Quantitative ultrastructural autoradiographic study of RNA transport in rat ventral prostate

The nucleocytoplasmic RNA transport in rat ventral prostate was studied by electron microscope autoradiography. Isolated prostate acini from normal, castrated, and DHT-treated animals were labeled in vitro with [³H]uridine for 5 min and chased for 0, 15, 30 min and 4 hr. The results show that (a) DHT induces a significant nucleolar enlargement but intranuclear migration of rRNA is not apparently affected by androgens; (b) migration of RNA through euchromatin is delayed by castration and stimulated by DHT; (c) migration through the nuclear envelope is androgen-dependent. In addition prostate acini were maintained for 24 hr in suspension culture in order to study the in vitro effects of DHT. The result show that (a) DHT stimulates uridine uptake and/or incorporation but induces no nucleolar enlargement; (b) DHT has no clear effects on RNA migration kinetics; (c) cytoplasmic transport of RNA in cells cultured in medium with or without DHT is severely impaired but is restored after supplementation of medium with insulin and dexamethasone.     

Nerve growth factor induced turnover of phosphatidylinositol in rat superior cervical ganglia

The addition of nerve growth factor to organ cultures of superior cervical ganglia from immature rats specifically stimulated the incorporation of 32P-orthophosphate into phosphatidylinositol fraction. Equimolar concentrations of other hormones such as insulin, glucagon, thyroxine and growth hormone did not cause any stimulation of the incorporation of 14C-myoinositol into phosphatidylinositol. The stimulation of phosphatidylinositol turnover was observed over a concentration of nerve growth factor ranging from 10^?10M to 10^?7M. Nerve growth factor specific “inositide effect” was found to be sensitive to nerve growth factor antibody, 2,4-dinitrophenol, a high concentration of bovine growth hormones but not to Actinomycin D. The physiological significance of this finding in relation to nerve growth factor action in this target tissue is discussed.     

Androgenic control of polysomal poly(A)-containing RNA in the cultured rat ventral prostate

Testosterone stimulated, at the concentration of 10-7 M and independently of other hormones, the accumulation of polysomal poly(A)-containing RNA (mRNA) in cultured explants of rat ventral prostate and concomitantly also protein synthesis. The hormone-induced accumulation of polysomal mRNA, which reached its maximum at 24 h after testosterone addition, paralleled the preferential labeling of high molecular weight RNA demonstrable with the electrophoretic analysis of the double-isotope labeled RNA after a short pulse (30 min). These findings are consistent with the idea that testosterone activated the synthesis of precursor mRNA leading to an increased amount of polysomal mRNA and eventually an activated protein synthesis. The synthesis and maturation of rRNA appeared to proceed even in the absence of testosterone, which is in contrast to the vivo findings on castrated rats. This partial uncoupling of RNA synthesis from androgenic control may account for the slow and less marked hormonal responses found in protein synthesis and glucose metabolism in cultured explants from normal animals. Because of the lack of uniformity in the suture, routine light microscopic control to assess the viability of cultured explants was found to be a prerequisite for successful biochemical work on prostate culture.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent snucleotide pools for nucleic acid synthesis

Novikoff rat hepatoma cells (subline NlSl-67) in suspension culture incorporate ³H-5-uridine into the acid-soluble nucleotide pool more rapidly than into RNA, resulting in the accumulation of labeled UTP in the cells. When labeled uridine is removed from the medium after 20 minutes or 4.75 hours of labeling, the rate of incorporation of label from the nucleotide pool into RNA decreases to less than 10% of the original rate within five to ten minutes, in spite of the presence of a large pool of labeled UTP in the cells, and incorporation ceases completely if an excess of unlabeled uridine is present during the chase. Upon addition of ¹⁴C-uridine to ³H-uridine pulse-labeled, chased cells, the ¹⁴C begins to be incorporated into RNA without delay and at a rate predetermined by the concentration of ¹⁴C-uridine in the medium and without affecting the fate of the free ³H-nucleotides labeled during the pulse-period. The results are interpreted to indicate that uridine is incorporated into at least two different pools, only one of which serves as primary source of nucleotides for RNA synthesis. During active synthesis of RNA, the latter pool of free nucleotides is very small and rapidly exhausted when uridine is removed from the medium. However, UTP accumulates in this pool when cells are labeled at 4–6°, since at this temperature RNA synthesis is blocked while uridine is still phosphorylated by the cells, and the UTP is rapidly incorporated into RNA during a subsequent ten-minute chase at 37°. From these types of experiments it is estimated that only 20–25% of the total uridine nucleotides formed in the cells from uridine in the medium is directly available for RNA synthesis and that the remainder becomes available only at a slow rate. Evidence is presented which suggests that one uridine nucleotide pool is located in the cytoplasm and another in the nucleus and that mainly the nuclear pool supplies nucleotides for RNA synthesis. The size of the latter pool is under strict regulatory control, since preincubation of the cells with 0.5 mM unlabeled uridine has little or no effect on the subsequent incorporation of ³H-uridine, although it results in an increase of the overall cellular uridine nucleotide content to at least 5 mM. Other results indicate that adenosine is also incorporated into two independent nucleotide pools, whereas the cells normally appear to possess a single thymidine nucleotide pool.     

A medium for the maintenance of Chironomus tentans salivary glands in vitro

A synthetic medium based upon the chemical composition of fourth instar Chironomus haemolymph was formulated for the in vitro culture of Chironomus tentans salivary glands.Salivary glands maintained in the medium for up to 4 days appeared morphologically normal. Secretion-free glands, obtained from pilocarpine-treated larvae, accumulated proteinaceous material in the gland lumen and exhibited a 46% increase in total gland protein after 24 hr in the medium. Cycloheximide almost totally inhibited the accumulation of secretion material and the increase in total gland protein by cultured glands.Glands cultured for up to 4 days continued to incorporate ¹⁴C-leucine into acid-insoluble total protein and ³H-uridine into total RNA, but at reduced levels. The incorporation of both isotopes was almost completely inhibited by cycloheximide.Autoradiographic squash preparations of glands pulse-labelled with ³H-thymidine after 3 days in culture revealed a normal pattern of asynchronous chromosomal DNA replication. Glands cultured for up to 4 days exhibited ³H-uridine incorporation into nucleoli and into distinct chromosomal regions which corresponded with sites of cytochemically demonstrable acidic protein.The chromosomes of cultured glands appeared morphologically and cytochemically normal, except for some regression of the Balbiani rings. Addition of ecdysterone to media containing glands previously cultured for 3 days resulted in puff induction at the IV-2-B chromosomal locus.     

Incorporation of label from radioactive uridine into the stylar nucleic acids of Lilium longiflorum thunb. as affected by heat, 6-methylpurine and actinomycin D

Summary 5-³H-uridine injected into the stylar canal of detached lily stigma-styles was taken up initially into the rapidly-labeled-RNA of the nucleic acid profile of a methylated albumin kieselguhr (MAK) column but with increasing time was found in all portions of the RNA profile, but not in the DNA. Heat treatment of the style before injection of 5-³H-uridine greatly reduced the rate of incorporation of label into and the ultimate amount of label found in the RNA species of the lily style. Translocation of 5-³H-uridine through the ovary into heattreated pistils and the injection of 5-³H-uridine into styles which had been incubated for 1 or 2 days after heat treatment resulted in stylar nucleic acids more highly labeled than nucleic acids in control styles, with an incorporation pattern different than control styles. Heat treatment of lily pistils resulted in detectable changes in the proportion of stylar RNA species as separated on MAK columns and measured as absorbance units. Actinomycin D and 6-methylpurine treated styles incorporated label from a stylar injection of radioactive uridine in patterns different than each other, different than heat-treated styles and different than non-treated styles. 6-methylpurine and heat treatment of styles only slightly reduced the rate at which 5-³H-uridine was removed from the stylar canal into the stylar tissue.Paper number 8917 of the Scientific Journal Series, Minn. Agr. Exp. Sta., St. Paul, MN 55108.     

Comparison of linoleate,palmitate and acetate metabolism in rat ventral prostate

Rat ventral prostate incorporated (1-¹⁴C)acetate, (1-¹⁴C)palmitate and (1-¹⁴C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI+LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+LPE. Acetate showed the highest oxidation to CO₂ whereas no differences were observed in the radioactivity incorporated into CO₂ from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO₂ oxidation.Abbreviations PL total phospholipids - PC choline glycerophospholipids - PE ethanolamine glycerophospholipids - PI+LPE inositol glycerophospholipids plus lysoethanolamine glycerophospholipids - PS serine glycerophospholipids - SM sphingomyelin     

Effect of light on ribonucleic Acid metabolism in greening maize leaves

The effect of illumination on the incorporation of labeled precursors into RNA of dark-grown maize (Zea mays) leaves was studied using either ³²P-phosphate or double labeling with ¹⁴C- and ³H-uridine. In the dark, label was preferentially incorporated into etioplast ribosomal RNAs. Incorporation into this fraction and into lower molecular weight fractions was strongly and preferentially stimulated by light during the first 2 hours of illumination. The effect persisted after illumination was terminated. The possibility that light-induced alterations in plastid ribosomal RNA metabolism may not be required for chlorophyll accumulation in maize is discussed.     

AN AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING POLLEN EMBRYOGENESIS IN HYOSCYAMUS NIGER (HENBANE)

RNA synthesis during pollen embryogenesis in cultured anther segments of Hyoscyamus niger (henbane) has been followed by autoradiography of ³H-uridine incorporation. Embryogenic divisions were initiated in binucleate pollen grains in which the generative nucleus or both generative and vegetative nuclei synthesized RNA. When the first haploid mitosis in culture resulted in pollen grains with two nearly identical nuclei, those in which both nuclei synthesized RNA became embryogenic. Binucleate pollen grains in which ³H-uridine incorporation was confined exclusively to the vegetative nucleus gradually became starch-filled and nonembryogenic. Based on the degree of involvement of the vegetative nucleus in embryoid formation, some differences were noted between the counts of autoradiographic silver grains over cells cut off by the generative and vegetative nuclei during progressive embryogenesis. The possible significance of RNA synthesis in the nuclei of binucleate pollen grains in determining the pathway of embryogenic divisions is discussed.     

Effects of 3′deoxyadenosine and actinomycin D on RNA synthesis in toad bladder: Analysis of response to aldosterone

Summary Previous studies have shown that aldosterone increases transepithelial active Na⁺ transport after a latent period of about 60 min and incorporation of³H-uridine into polyadenylated RNA (poly(A)(+)RNA) (putatively poly(A)(+)mRNA) as early as 30 min after aldosterone addition. To assess the physiological importance of this pathway, the effects of 3deoxyadenosine and actinomycin D were compared in studies on the urinary bladder of the toadBufo marinus. 3deoxyadenosine (30 g/ml) only partially, though significantly, inhibited the aldosterone-dependent increase in Na⁺ transport measured as short-circuit current (scc). The incorporation of³H-uridine into poly(A) (+)RNA was inhibited by 70 to 80%. In contrast, Actinomycin D (2 g/ml) totally inhibited the aldosterone-dependent increase in scc, and the incorporation of³H-uridine into poly(A)(+)RNA by 68 to 75%. 3deoxyadenosine or actinomycin D alone had no significant effects on baseline scc, while inhibiting poly(A)(+)RNA to the same extent. The differential effects of deoxyadenosine and actinomycin on aldosterone-dependent Na⁺ transport may be related to their different sites of action on RNA synthesis: both drugs inhibited, to a similar extent, cytoplasmic poly(A)(+)mRNA; however, 3deoxyadenosine, in contrast to Actinomycin D, failed to inhibit poly(A)(-)RNA, sedimenting between 4S and 18S (putatively poly(A)(-)mRNA). We conclude that the mineralocorticoid action of aldosterone during the first three hours depends on the synthesis of both poly(A)(+)mRNA and poly(A)(-)mRNA.     

The effect of synaptic stimulation on RNA and protein metabolism in the R2 soma of Aplysia

Excitatory synaptic stimulation of the R2 neuron in the abdominal ganglion of Aplysia californica causes an increased incorporation of ³Huridine into RNA. However, this could be the result of a change in precursor specific activity rather than an increase in RNA synthesis. We find that at low external uridine concentrations (1.5 μM) there is no increase in ³H-uridine incorporation correlated with synaptic stimulation. In addition, no change in incorporation of ³H-leucine into total protein or in the pattern of newly-synthesized proteins, resolved by electrophoresis on SDS-polyacrylamide gels, was detected with stimulation. Since the R2 neuron can be stimulated without a detectable change in RNA or protein synthesis, we conclude that the increase in incorporation observed at high external uridine concentrations (100 μM) could be caused by increased specific activity in a precursor pool rather than by an RNA synthesis change.     

A novel nuclear protein in rat ventral prostate androgen-dependent and age-related change

A novel protein was found in the nuclei of rat ventral prostate. This protein has a molecular weight of about 21 kDa as measured by SDS-polyacrylamide gel electrophoresis. It showed a characteristic change between 3 and 84 weeks after birth in close association with the level of testosterone in the blood. After castration, the level of the 21-kDa protein decreased to $\text{1}\text{60}$ of normal in 7 days, but on daily injection of testosterone the level was restored to normal in 8 days and to twice the normal level in 14 days. Unlike H₁ and H₁⁰ histone and high mobility group proteins, the 21-kDa protein was not extracted with 5% HClO₄, but was partially extracted with 0.35 M NaCl. The 21-kDa protein was not found in kidney, liver, or brain, suggesting that it is specific to the ventral prostate.     

Inhibition of polyadenylation of mRNA by gonadotropin releasing hormone

The mechanism of extra pituitary inhibitory action of gonadotropin releasing hormone (GnRH) was investigated. Simultaneous injection of GnRH caused dose dependent inhibition of dihydrotestosterone (DHT) induced poly(A) polymerase in the ventral prostate of rat. In addition injection of GnRH to DHT treated animals caused reduced incorporation of 3H-uridine into poly(A)+ mRNA. Since poly(A) segment is known to help in translation of mRNA, it is possible that the inhibitory effect of GnRH is due to the inhibition of polyadenylation of mRNA.