Effect of hyperphenylalaninaemia on lipid synthesis from ketone bodies by rat brain.

The effect of hyperphenylalaninaemia on the metabolism of ketone bodies in vivo and in vitro by developing rat brain was investigated. The incorporation in vivo of [14C]acetoacetate into cerebral lipids was decreased by both chronic (for 3 days) and acute (for 6h) hyperphenylalaninaemia induced by injecting phenylalanine into 1-week-old rats. In studies in vitro it was observed that the incorporation of the radioactivity from [14C]acetoacetate and 3-hydroxy[14C]butyrate into cerebral lipids was inhibited by phenyl-pyruvate, but not by phenylalanine. Phenylpyruvate also inhibited the incorporation of 3H from 3H2O into lipids by brain slices metabolizing either 3-hydroxybutyrate or acetoacetate in the presence of glucose. These findings suggest that the decrease in the incorporation in vivo of [14C]acetoacetate into cerebral lipids in hyperphenylalaninaemic rats is most likely caused by phenylpyruvate and not by phenylalanine. Phenylpyruvate as well as phenylalanine had no inhibitory effects on ketone-body-catabolizing enzymes, namely 3-hydroxybutyrate dehydrogenase, 3-oxo acid CoA-transferase and acetoacetyl-CoA thiolase, in rat brain. Phenylpyruvate but not phenylalanine inhibited the activity of the 2-oxoglutarate dehydrogenase complex from rat and human brain. These findings suggest that the metabolism of ketone bodies is impaired in brains of untreated phenylketonuric patients, and in turn may contribute to the diminution of mental development and function associated with phenylketonuria.     

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.     

Inhibition by warfarin of prothrombin synthesis and of lipid-saccharide synthesis in rat liver

In vivo and in vitro studies using [³H]glucosamine incorporation into prothrombin and into glycolipids were conducted in rat liver to determine the role of lipid-saccharides in the biosynthesis of prothrombin.In vivo studies demonstrated that 10 mg warfarin/kg inhibited the incorporation of radiolabeled glucosamine into liver prothrombin and glycolipids. This inhibition was similar to the kinetics of inhibition of prothrombin synthesis in the liver.In vitro studies demonstrated a time-dependent increase in the incorporation of radiolabeled glucosamine into lipid-saccharides and prothrombin. This incorporation was inhibited 50% by 5 · 10^?4 M warfarin. Warfarin also inhibited the incorporation of radiolabeled glucosamine into glycolipids in a dose-related manner.In all studies, vitamin K-1 reversed the inhibition of glucosamine incorporation into glycolipids and into prothrombin.     

In vivo synthesis and processing of rat hypothalamic prosomatostatin

The in vivo incorporation of [3H]phenylalanine into an apparent 15 kDa prosomatostatin was observed in the hypothalamus of rats injected with the labeled amino acid in the third ventricle. Precursor-product relationships were established between this newly synthesized material and both somatostatin-28 and -14.     

Calcium-dependent inhibition of protein synthesis in rat parotid gland.

1. Protein synthesis in the rat parotid gland in vitro was studied by measuring the incorporation of [3H]phenylalanine into trichloroacetic acid-insoluble proteins. In the unstimulated gland, the rate of incorporation was dependent on the phenylalanine concentration in the medium and proceeded linearly for up to 3h. 2. Adrenaline, carbamoylcholine, phenylephrine and ionophore A23187 inhibited the incorporation of [3H]phenylalanine into acid-insoluble protein; isoprenaline, dibutyryl cyclic AMP and 8-bromo-cyclic GMP were inactive. 3. Inhibition by adrenaline and carbamoylcholine but not by ionophore A23187 required extracellular Ca2+. 4. Both adrenaline and carbamoylcholine increased the magnitude of the acid-soluble [3H]phenylalanine pool at 10 micrometer extracellular phenylalanine, but had no effect if the phenylalanine concentration was increased to 200 micrometer. 5. There was no correlation between cellular ATP content and the observed inhibition of protein synthesis. 6. Our results suggest that both alpha-adrenergic and cholinergic receptors may play a role in the regulation of protein synthesis in the rat parotid gland, and that their effects are mediated by a rise in intracellular free Ca2+.     

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 Km and V in the incorporation of aromatic amino acids into protein and amino acyl-tRNAs, even if V decreased more than Km.     

Effect of excessive vitamin A intake on muscle protein turnover in the rat.

3-Methylhistidine excretion in vivo and in vitro was monitored in hypervitaminotic and pair-fed control rats. Feeding with excess of retinyl palmitate (40 000 i.u./day per 100 g body wt.) significantly increased urinary 3-methylhistidine and creatinine output during a 4-day treatment interval. 3-Methylhistidine release from perfused rat hindquarters was also elevated after 5 days of vitamin treatment. To determine whether the adrenals were involved in mediating the above response, a study was conducted on adrenalectomized and sham-operated rats. Excessive vitamin A intake stimulated 3-methylhistidine excretion in vivo and in vitro in both adrenalectomized and sham-operated animals, thus suggesting that the vitamin A-induced acceleration in myofibrillar protein breakdown was not mediated by the adrenals. In both groups of rats, vitamin A treatment had no effect on the rate of protein synthesis, on the basis of incorporation in vitro of [3H]phenylalanine into muscle protein. Additional studies revealed that the addition of excess retinol to the perfusion medium (10 i.u./ml) had no significant effect on the rates of 3-methylhistidine release or [3H]phenylalanine incorporation in vitro. Finally, high doses of cortisol (7 mg/day per 100g body wt.) administered to intact rats for 5 days significantly increased rates of 3-methylhistidine excretion, both in vivo and in vitro.     

Impairment of pancreatic acinar function by reserpine in vivo and in vitro

Summary Chronic reserpine treatment of animals, an experimental model for cystic fibrosis (CF), results in generalized exocrinopathy, impaired pancreatic secretion, and decreased pancreatic content of amylase. The mechanisms of altered acinar function and decreased amylase content in both CF and the reserpine-treated rat are unknown. To examine this alteration, the rate of [³H]phenylalanine (phe) incorporation into cellular protein was determined in pancreatic acinar cells after reserpine treatment of rats in vivo (7 d) and of cells in vitro (1 to 24 h). Acinar cells isolated from control, chronic reserpine-treated, and pair-fed rats were incubated in vitro with 0, 30, 50, or 100 μM reserpine. Reserpine treatment in vitro for 24 h of acinar cells from control rats significantly decreased amylase activity (20 to 70%), an effect similar to that of reserpine treatment in vivo. In vivo, reserpine treatment decreased [³H]phe incorporation (disintegrations per minute per milligram protein) 56% in freshly isolated cells, but did not alter intracellular specific activity (disintegrations per minute per nanomole phe, SA) of [³H]phe. Reserpine treatment (30 and 50 μM) in vitro for 1 h also decreased [³H]phe incorporation by freshly isolated cells from control (53 to 85%) and pair-fed (40 to 68%) rats. Reserpine treatment for 24 h in vitro significantly decreased [³H]phe incorporation by cells from control (82 and 98%), pair-fed (80 and 95%), and chronic reserpine-treated (90 and 97%) rats as compared with cells from respective in vivo treatments cultured with no reserpine. In vitro reserpine treatment also decreased the intracellular SA of [³H]phe in freshly isolated cells from control (14 and 36%) and pair-fed (35 and 39%) rats and in cultured cells from control (11 and 86%), pair-fed (60 and 88%), and chronic reserpine-treated (49 and 76%) rats. However, these alterations of SA by reserpine did not account for the decreased incorporation of [³H]phe into acinar protein, which remained significantly lower (70 to 88%) when expressed as total phe incorporation. These results suggest (a) that reserpine acts directly on acinar cells to alter function and (b) that the ability of the pancreas to synthesize digestive enzymes may be impaired in this model of cystic fibrosis. This study was supported in part by the Cystic Fibrosis Foundation, Bethesda, MD.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Increased Fucosylation of Chick Brain Proteins Following Training: Effects of Cycloheximide

When chicks are trained to avoid pecking a bead coated with methylanthranilate in a one-trial passive avoidance task there is an increase in fucose incorporation in vivo and in vitro in the right forebrain base of methylanthranilate (M)-trained compared to water (W)-trained chicks. The relation of this increase to de novo protein synthesis in vivo and in vitro has been examined. Cycloheximide (Cx), 1 mM, inhibited in vitro fucosylation of chick brain slices by 60% after 3 h. However, the training-related increase in in vitro fucosylation still persisted. When Cx was injected intraventricularly 10 min before training, the subsequent increase in in vitro fucosylation due to training was still apparent. When Cx was injected and [14C]leucine and [3H]fucose incorporation studied in vivo in M-trained and W-trained chicks, there was no increase in fucosylation due to training in the Cx-treated M-trained over the W-trained chicks. These results are taken to indicate that in vitro fucosylation and its increase subsequent to training is not protein synthesis-dependent, but that both in vivo and in vitro there are interactions between Cx and fucosylation steps that are independent of Cx's effects on protein synthesis.     

The effect of oophorectomy on the ribonucleic acid metabolism of a transplantable uterine tumour

1. The incorporation of [(14)C]phenylalanine and of [(3)H]uridine into the Guerin T8 uterine carcinoma has been examined. 2. The addition in vitro of polyuridylic acid caused an increase in the incorporation of labelled phenylalanine into tumour microsomes obtained from both oophorectomized and intact animals. 3. Oophorectomy of tumour-bearing animals decreased the specific activity due to the uptake in vivo of [(3)H]uridine into both the soluble and insoluble RNA of the tumour. 4. Actinomycin D caused a decrease in specific activity of RNA in the tumour in vivo, and puromycin inhibited uptake in vitro into tumour microsomes.     

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.     

Phospholipase A2 Stimulation by Methyl Mercury in Neuron Culture

Abstract: In primary prelabeled cultures of cerebellar granule cells, methyl mercury (MeHg) induced a concentration- and time-dependent release of [³H]arachidonic acid. MeHg-induced [³H]arachidonate release was partially dependent on the extracellular Ca²⁺ concentration. MeHg at 10–20 µM also stimulated basal ⁴⁵Ca²⁺ uptake after 20 min of incubation at 37°C, and at 10 µM inhibited K⁺ depolarization-stimulated uptake. MeHg stimulated [³H]arachidonate uptake, but had no effect on the rate of phospholipid reacylation. Phospholipase A₂ (PLA₂) activation preceded cytotoxicity, but at higher concentrations of MeHg such dissociation was not evident. Inhibition of MeHg-induced PLA₂ activation by 100 µM mepacrine failed to modify cytotoxicity. MeHg-induced lipoperoxidation, measured as the production of thiobarbituric acid-reacting products, was inhibited by α-tocopherol without inhibition of [³H]arachidonate release. The absence of α-tocopherol inhibition of MeHg-induced arachidonate release precludes a causal role for lipoperoxide-induced PLA₂ activation in this system. Moreover, MeHg induced an increased susceptibility of unilamellar vesicles to exogenous PLA₂ in the presence of low Ca²⁺ concentrations without evidence of lipid peroxidation. [³H]Arachidonate incorporation into granule neuron phospholipids was analyzed by isocratic HPLC analysis. Relatively high proportional incorporation was found in the combined phosphatidylcholine fractions and phosphatidylinositol. With MeHg, an increase in the relative specific activity of incorporation was found in the phosphatidylinositol fraction, indicating a preferential turnover in this phospholipid species in the presence of MeHg.     

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.     

EFFECTS OF AMPHETAMINE ADMINISTRATION IN VIVO ON IN VITRO PROTEIN SYNTHESIZING SYSTEM FROM RAT BRAIN 1

Abstract— A highly active in vitro protein synthesizing system (S-28) has been prepared from rat brain. Poly (U)-dependent [³H] phenylalanine incorporation by brain S-28 system is significantly inhibited by D-amphetamine. The extent of inhibition by amphetamine is significantly higher than by other biogenic amines such as dopamine and serotonin. At the 100°g level of amphetamine, the inhibition is about 70°. Experiments with ribosomes and soluble enzymes from control and amphetamine-treated systems indicate that the observed inhibition may be due to the effect of the drug on the ribosomes. Kinetic analysis of the reaction mixture in the presence as well as absence of D-amphetamine indicate that this sympathomimetic drug inhibits polysome formation in vitro.     

Biochemical and Autoradiographic Study of Cerebral Protein Synthesis with [18F]- and [14C]Fluorophenylalanine

Fluorine-18-labeled ortho or para isomers of L-fluorophenylalanine were used in double-label experiments together with L-[3H]phenylalanine for amino acid incorporation into cerebral proteins of Mongolian gerbil brain. It was demonstrated by qualitative regional comparison of the 18F and 3H autoradiographic images that L-p-[18F]fluorophenylalanine is incorporated into proteins and exhibits a regional cerebral protein synthesis pattern. To a minor extent, L-p-fluorophenyl[3-14C]alanine and L-o-[18F]fluorophenylalanine are hydroxylated in vivo to form labeled tyrosine or tyrosine analogues that are incorporated into cerebral proteins as well. The advantage and validity of the application of L-p-[18F]fluorophenylalanine with positron emission tomography for noninvasive studies of cerebral protein synthesis in humans are evaluated on the basis of an experimental animal approach.     

Biosynthesis of liver catalase in rats treated with allylisopropylacetylcarbamide. II. Double-labeling of catalase with (14C)leucine and delta-(3H)aminolevulinic acid.

Double-labeling of liver catalase [EC 1.11.1.6] with [14-c]leucine and delta-[3H]aminolevulinic acid was carried out both in vivo and in vitro using rats treated with allylisopropylacetylcarbamide (Sedormid). These radioactive precursors were incorporated into catalase at a lower rate than in normal rats. In particular, the incorporation of 3H was remarkably inhibited. The results suggest that the administration of Sedormid can inhibit synthesis of the protein moiety of catalase, and possibly interfere with the binding of heme to the catalase protein.     

Effect of phenylalanine on protein synthesis in the developing rat brain

1. Inhibition of the rate of incorporation of [(35)S]methionine into protein by phenylalanine was more effective in 18-day-old than in 8-day-old or adult rat brain. 2. Among the subcellular fractions incorporation of [(35)S]methionine into myelin proteins was most inhibited in 18-day-old rat brain. 3. Transport of [(35)S]methionine and [(14)C]leucine into the brain acid-soluble pool was significantly decreased in 18-day-old rats by phenylalanine (2mg/g body wt.). The decrease of the two amino acids in the acid-soluble pool equalled the inhibition of their rate of incorporation into the protein. 4. Under identical conditions, entry of [(14)C]glycine into the brain acid-soluble pool and incorporation into protein and uptake of [(14)C]acetate into lipid was not affected by phenylalanine. 5. It is proposed that decreased myelin synthesis seen in hyperphenylalaninaemia or phenylketonuria may be due to alteration of the free amino acid pool in the brain during the vulnerable period of brain development. Amyelination may be one of many causes of mental retardation seen in phenylketonuria.     

Lack of effect of propylthiouracil and methylmercaptoimidazole on thyroglobulin biosynthesis

Experiments were performed both in vivo and in vitro to test a previous proposal that part of the antithyroid action of the thioureylene drugs, propylthiouracil (PTU) and methylmercaptoimidazole, can be attributed to inhibition of thyroglobulin (Tg) biosynthesis. Rat thyroid lobes were incubated in leucine-free Eagle's medium containing bovine thyroid-stimulating hormone and 0, 0.1-0.2, or 1 mM drug. After a 30-min preincubation, 5 mu Ci of [14C]leucine were added and the incubation was continued for 4 hr. The soluble fraction was analyzed by sucrose density gradient centrifugation, and the fractions corresponding to the 19S Tg peak were pooled and assayed for 14C. No inhibition of 14C incorporation into 19S Tg was observed, even in thyroid lobes incubated in the presence of 1 mM methylmercaptoimidazole or 2 mM PTU. At the same time, 14C incorporation into 19S Tg was completely inhibited when lobes were incubated in the presence of 0.1 mM puromycin. In vivo, rats received an injection of PTU (1 mumol/100 g body wt), followed 60 min later by an injection of 25 mu Ci of [14C]leucine. Blood samples and thyroids were taken 5 hr after the [14C]leucine injection. Serum thyroid-stimulating hormone was not significantly affected by the PTU injection. The thyroid-soluble fraction was analyzed by sucrose density gradient centrifugation. No significant differences between saline and PTU-injected groups were observed in [14C]leucine incorporation into 19S Tg. We conclude from both our in vitro and our in vivo studies that PTU and methylmercaptoimidazole have no inhibitory effect on thyroglobulin synthesis in rat thyroids and that such inhibition does not play a significant role in the antithyroid action of these drugs.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.