Polypeptide Synthesis with Microsomes from Pressure-Treated Tetrahymena1

SYNOPSIS High hydrostatic pressure is known to interfere with mitosis, cytokinesis and synthesis of DNA, RNA and protein. In Tetrahymena, incorporation of phenylalanine and formation of polysomes are known to be pressure-sensitive. Microsomal preparations from Tetrahymena pyriformis GL can incorporate [¹⁴C]-phenylalanine into polypeptides. Incorporation was enhanced by addition of supernatant fraction and 14.5 mM Mg⁺⁺ An energy-generating system and exogenous messenger (poly U) were essential for polypeptide biosynthesis, Microsomes from pressurized cells (14,000 psi for 5 min) incorporated [¹⁴C]phenylalanine as efficiently as control microsomes. Microsomal function was not grossly damaged by pressure in a test system containing exogenous messenger, crude microsomal preparation, exogenous energy-generating system and supernatant fraction containing activating enzymes.     

Inhibition of conjugation and cell division in Tetrahymena pyriformis by tunicamycin: A possible requirement of glycoprotein synthesis for induction of conjugation

Tunicamycin, a glucosamine-containing antibiotic inhibited the conjugation process of Tetrahymena pyriformis. Sexual pairing was prevented completely when 1.5 μg/ml of tunicamycin was added to a mixture of the two mating types. Tunicamycin caused preferential inhibition of glycoprotein synthesis in Tetrahymena pyriformis. At 1.5 μg/ml and 6 μg/ml tunicamycin inhibited by 40% and 60% respectively [³H]-glucosamine incorporation into material precipitated by ethanol, while it did not affect [¹⁴C]-leucine incorporation. Cell division was also inhibited when the drug was added either to the regular growth medium or to the starvation medium.     

The action of 5-amino uracil on log growth and division-synchronized Tetrahymena

The influence of 5-amino uracil (5-AU) was investigated on the cell cycle of log growth and division-synchronized Tetrahymena pyriformis GL. The division index of log growth phase Tetrahymena was suppressed by 50% after 40 min in 8 mM 5-AU. Cells division-synthronized by one heat shock per generation were also treated with 5-AU. Cells treated either prior to the first synchronous division (80 min EH) or up to 25 min prior to the second synchronous division (after 160 min EH) were not delayed in their progress through the cell cycle. Cells treated during the S phase of the first free running cell cycle, however, were delayed 5-30 min from reaching the second synchronous division. The effect of 5-AU on DNA and RNA synthesis was also examined. Incorporation of [3H]thymidine into acid-precipitable material was reduced in the presence of 5-AU; the rate of DNA synthesis was also reduced. The depression in the rate of DNA synthesis was greater at the beginning of S than at the end of S. The size of the thymidine pool (nucleosides + nucleotides) did not change during 5-AU treatment; however, an accumulation of thymidine tri-phosphate and a decrease in the amount of thymidine nucleoside was observed. A suppression of [14C]uridine incorporation resulting from 5-AU treatment was observed throughout the cell cycle. The rate of RNA synthesis as monitored by [14C]uridine incorporation into acid precipitable material was also reduced during 5-AU treatment. No change in either the size or the composition of the pool of uridine (nucleoside + nucleotide) was detected in 5-AU treated cells as compared to controls.     

The Action of Mercaptoethanol on Cell Division in Synchronized Tetrahymena1

SYNOPSIS. Action of mercaptoethanol (ME) on cell division and macromolecular synthesis was examined in Tetrahymena synchronized for division. Cells continuously exposed to increasingly higher concentrations of ME divided with progressively longer division delays showing a dosage-dependent response to the agent. Division was blocked in 2 × 10^?2 M ME. Many cells cytolyzed in high concentrations of ME (4 × 10^?2 M); others became spherical and motility decreased. Non-delaying concentrations of ME (2 × 10^?2 M) had little or no effect on protein synthesis but decreased DNA and RNA synthesis 10 and 35%, respectively. Blocking concentrations inhibited incorporation of phenylalanine, thymidine and uridine 35, 60, and 85%, respectively. It is suggested that the mode of action of ME is mediated thru inhibition of macromolecular synthesis essential for cell division and thru inhibition of formation of disulfide bridges between protein subunits.     

Microcyst germination in the cellular slime mold, Polysphondylium pallidum: Effects of actinomycin D and cycloheximide on macromolecular synthesis and enzyme accumulation

Germination of microcysts of Polysphondylium pallidum is characterized by an immediate rapid increase in incorporation of [³H]leucine into protein which is cycloheximide-sensitive but unaffected by actinomycin D. Significant RNA synthesis, as measured by [³H]uridine incorporation, does not begin until approx. 2 h after the onset of germination. The increase in [³H]uridine incorporation is prevented by actinomycin D. Germination and the increase in alkaline phosphatase and β-glucosidase enzyme activities are prevented by cycloheximide but unaffected by actinomycin D. The data strongly imply the presence of stable RNA in dormant microcysts and indicate a requirement for a discrete period of protein synthesis for germination of microcysts of P. pallidum.     

Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [³H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [³H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [³H]uridine by > 75%. Cellular uptake of [³H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [³H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.     

Manipulation,by nutrient limitation,of the biosynthetic activity of immobilized cells of Capsicum frutescens Mill. cv. annuum

The relationship between the synthesis and accumulation of protein and capsaicin was investigated in cultured cells of Capsicum frutescens Mill. cv. annuum immobilized in reticulate polyurethane. Cells were cultured in media containing reduced concentrations of essential nutrients, in an attempt to manipulate the rates of protein synthesis. Cells cultured in the absence of orthophosphate for 7 d demonstrated no reduction in the incorporation of l-[U-¹⁴C]phenylalanine into soluble protein or an increase in incorporation into capsaicin, compared with controls supplied with orthophosphate. By day 15 of culture, however, a differential incorporation of label was observed. Over a 21-d culture period the intracellular phosphate did not completely disappear. Cells cultured in the absence of nitrate and phosphate combined, however, exhibited some reduction in incorporation of [¹⁴C]phenylalanine into protein and an increased incorporation into capsaicin after 7 d of culture, but the differences were greater at day 15, when increases in the total capsaicin content of the cultures were apparent. There was observed a relationship between the intracellular nitrate concentration, the culture growth index, and the incorporation of [¹⁴C]phenylalanine into soluble protein — each of these factors was inversely related to the incorporation of label into capsaicin and the total capsaicin content of the cultures.Abbreviations HPLC high-performance liquid chromatography - Phe phenylalanine     

RNA synthesis during spermiogenesis in the sea urchin: a high resolution autoradiographic study of [3H]uridine incorporation

Summary

RNA synthesis has been studied during spermiogenesis of Paracentrotus lividus by high resolution autoradiography using [³H]uridine as a labeled precursor. Under the experimental conditions used [³H]uridine incorporation is detectable only at the early spermatid stage. Labeling is distributed mainly over the nucleus and it appears completely absent from the mitochondria. After RNase digestion radioactivity falls to a background level, demonstrating that RNA synthesis actually occurs in early spermatids of P. lividus. It follows that in P. lividus during spermiogenesis cell differentiation is not entirely dependent on stored premeiotic RNAs.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

Inhibition of nucleic acid synthesis in Ehrlich tumor cells by periodate-oxidized adenosine and adenylic acid

Periodate-oxidized adenosine and AMP were inhibitory to both RNA and DNA synthesis in Ehrlich tumor cells in culture. With periodate-oxidized adenosine, the inhibition of RNA synthesis paralleled the inhibition of DNA synthesis. Periodate-oxidized AMP, however, was more inhibitory to DNA synthesis than to RNA synthesis. With both compounds, there was a decrease in the conversion of [¹⁴C]cytidine nucleotides to [¹⁴C]deoxycytidine nucleotides in the acid-soluble pool. The borohy-dride-reduced trialcohol derivative of the periodate-oxidized adenosine compound was not inhibitory to DNA or RNA synthesis in the tumor cells. The incorporation of [³H]uridine into 28S and 18S ribosomal RNA was inhibited by both periodate-oxidized adenosine and AMP, but the incorporation of [³H]uridine in 45S, 5S, and 4S RNA was essentially unaffected by these compounds. Periodate-oxidized adenosine inhibited Ehrlich tumor cell growth in vivo.     

Effect of aromatic acids on protein synthesis in subcellular preparations from the rat brain

The incorporation of [³H]phenylalanine, [³H]tyrosine, and [³H]tryptophan into protein and amino acyl–tRNA was studied in cell-free preparations from rat brain. Tyrosine and tryptophan inhibited the incorporation of phenylalanine into protein, and tyrosine inhibited the incorporation of phenylalanine and tryptophan into amino acyl–tRNAs. In most cases, homogentisate, phenylpyruvate, and phenyllactate inhibited the incorporation of phenylalanine, tyrosine, and tryptophan into protein and amino acyl–tRNAs, and the incorporation of phenylalanine into polyphenylalanine. All other protein amino acids, and phenylacetate, salicylate, and benzoate were wholly ineffectual. The results suggest that the formation of amino acyl–tRNAs may have been the step which was affected most by the inhibitors. The incorporation data at different concentrations of the aromatic amino acids were fitted to the simple Michaelis equation. Homogentisate and phenylpyruvate generally tended to reduce both K_m and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than K_m.     

An autoradiographic method of detectingA. laidlawii andM. hyorhinis in cell cultures

Summary The autoradiographic investigation of L cells and Chinese hamster cells for the presence of mycoplasmas (A. laidlawii andM. hyorhinis) using uridine/uracil (UdR/U) testing is a rapid and reliable method suitable for the serial checking of even a small number of cells. It depends on a reduced incorporation of [³H]uridine and an increased uptake of [³H]uracil into the RNA of mycoplasma-infected cells, shown in autoradiograms by the density of the grains and their distribution. Results obtained by the autoradiographic technique correspond approximately to specific activity values of RNA-infected cells after the incorporation of [³H]uridine and [³H]uracil.     

INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

Effect of garlic oil on incorporation of amino acids into proteins of Culex pipiens quinquefasciatus Say larvae

Incorporation of [¹⁴C]leucine into proteins of 3rd instar Culex pipiens quinquefasciatus Say larvae increases linearly with time between 1 and 4 h. Garlic oil as well as the active larvacidal principle from it, viz. diallyl disulphide, inhibits significantly synthesis of the larval proteins. The maximum reduction in incorporation is observed during the first hour of treatment. The incorporation of [¹⁴C]phenylalanine is also inhibited by garlic oil and the effect is irreversible. Garlic oil does not seem to have any effect on proteins already labelled and it does not suppress substantially oxygen uptake by the larvae.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

EFFECTS OF ADDED SUBSTANCES ON RNA AND PROTEIN SYNTHESIS IN IMMATURE NEURONS

Abstract— A newly described method for the isolation of morphologically intact neurons from newborn rat brain was used to study the influence of inhibitors and neuroactive substances on RNA and protein synthesis in these cells in vitro . Incorporation of [¹⁴C]-uridine into RNA and [³H]leucine into protein proceeded rapidly and continued up to 3 h. When the incorporation mixture was chased at 20 min with an excess of nonradioactive uridine and leucine, hardly any degradation of labelled RNA was noted during the following 2 h 40 min. In contrast, the specific radioactivity of proteins decreased by 22 per cent indicating turnover of cellular proteins.
Incorporation of labelled leucine into protein was markedly inhibited in the presence of NaF and cycloheximide but not affected in the presence of chloramphenicol or pancreatic RNase. A mixture of ATP + GTP depressed the incorporation by 38 per cent. The responses to ATP + GTP and RNase indicated that the incorporation system was typically cellular. Acetylcholine, γ-aminobutyrate, noradrenaline and phenylalanine in the incubation medium depressed the incorporation of labelled uridine into RNA by 10–30 per cent and 5-hydroxytryptamine by 75 per cent. Acetylcholine, γ-aminobutyrate and noradrenaline had no effect on protein synthesis, while 5-hydroxytryptamine and phenylalanine inhibited the incorporation by 60–80 per cent. Testosterone and prednisolone depressed both RNA and protein synthesis while thyroxine caused slight but non-significant stimulation.     

Effect of hypoxia on nucleic acid and protein synthesis in different brain regions

The incorporation of [methyl-³H]thymidine into DNA, of [5-³H]uridine into RNA, and of [1-¹⁴C]leucine into proteins of cerebral hemispheres, cerebellum, and brainstem of guinea pigs after 80 hr of hypoxic treatment was measured. Both in vivo (intraventricular administration of labeled precursors) and in vitro (tissue slices incubation) experiments were performed. The labeling of macromolecules extracted from the various subcellular fractions of the above-mentioned brain regions was also determined. After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the three brain regions examined were particularly affected.