The effect of actinomycin D on the synthesis of ribonucleic acid and protein in rat liver parenchymal cells in suspension and liver slices

1. Rat liver parenchymal cells in suspension are shown to require a higher concentration of actinomycin D than liver slices for equivalent inhibition of the incorporation of [(14)C]adenine, [(14)C]uracil and [(32)P]phosphate into RNA, and of (14)C-labelled amino acids into protein; protein synthesis is much less susceptible to actinomycin D inhibition than RNA synthesis in both the tissue preparations. Possible causes for these differences are discussed. 2. The uptake of [(3)H]actinomycin D in the first few minutes was much greater in the cell suspensions than in the tissue slices; that in the next 1-4hr. was about the same in both the cases. The uptake by both the tissue preparations was at all times proportional to the concentration of the drug within the range 0.5-2.0mug./ml. 3. In the slices actinomycin D taken up initially was concentrated almost exclusively in the nuclei; with time the concentration of the drug in the mitochondria and the supernatant increased more rapidly than in the nuclei though at no stage did it exceed that in the nuclei. In the cell suspension the largest concentration of the drug taken up initially was found in the supernatant; most of the drug taken up subsequently also stayed in the supernatant. 4. When the drug concentration in the incubation medium was 1mug./ml., its concentration within the parenchymal cells in suspension and the parenchymal cells in the slices reached 2.2 and 1.6mug./cm.(3) of cellular volume respectively. On average, 7% of the drug was removed from the medium by the cells in suspension and 23% by the cells in the slices; the average ratio of intracellular to extracellular concentration was 2.4 in the former and 2.1 in the latter case.     

The utilization of ketone bodies by the interscapular brown adipose tissue of the rat

The activities of 3-oxo acid-CoA transferase (EC 2.8.3.5, 13-15 micromol/min per g) and acetoacetyl-CoA thiolase (EC 2.3.1.9, 18-21 micromol/min per g) in interscapular brown adipose tissue of the rat are comparable to the activities reported for heart and kidney. The incorporation of D-3-hydroxy[3-14C]butyrate into lipid in vivo was about 30-fold higher in interscapular brown adipose tissue than in white adipose tissue of virgin rats. In lactating rats, the mammary gland was the major site of ketone body incorporation into lipid and incorporation of D-3-hydroxy-[3-14C]butyrate into lipid in brown adipose tissue was lower than in virgin rats. After an oral load of medium chain triacylglycerol, which inhibits lipogenesis in lactating mammary gland, the incorporation of ketone bodies into lipid was decreased in mammary gland but increased in brown adipose tissue. The rate of oxidation of D-3-hydroxy[3-14C]butyrate by brown adipose tissue slices in vitro was higher than the rate of incorporation into lipid.     

The protein-synthesizing activity of ribosomes isolated from the mammary gland of lactating and pregnant guinea pigs

1. Polyribosome preparations were made from the deoxycholate-treated post-nuclear fractions obtained by the disruption of mammary glands from lactating and pregnant guinea pigs. 2. A high proportion of large polyribosomes was obtained from the glands of lactating animals whereas mainly small polyribosomes were obtained from the glands of pregnant animals. The isolated preparations incorporated [(14)C]phenylalanine into protein. The polyribosomes from the glands of pregnant animals were less active than those from the glands of lactating animals but the activity of the former was stimulated more by poly(U) than was the latter. 3. The ribosomes from mammary gland could be dissociated into subunits after incubation, under conditions necessary for protein synthesis, in the presence of puromycin. The subunits could be recombined to give a preparation that actively polymerized [(14)C]phenylalanine in the presence of poly(U). The subunits from guinea-pig mammary gland could be combined with subunits from liver of either guinea pig or rat. Hybrid ribosomes were also formed from subunits derived from glands of pregnant and lactating animals. The hybrids were as active as were the ribosomes formed by reassociation of subunits from the same tissue, suggesting that in this respect the ribosomes from pregnant animals were not defective. 4. Polyribosomes from mammary glands of lactating animals when incubated with cell sap from the same source were tested for their ability to synthesize alpha-lactalbumin. The polyribosomes were incubated in the presence of [(3)H]leucine and alpha-lactalbumin was isolated from the supernatant. The protein was finally treated with cyanogen bromide and the C-terminal and N-terminal fragments were separated and their radioactivity was determined. Both fragments were radioactive consistent with the synthesis of alpha-lactalbumin. 5. The results are discussed in relation to protein synthesis in the mammary gland after parturition.     

Polymyxin B diminishes blood flow to brown adipose tissue and lactating mammary gland in the rat. Possible mechanism of its action to decrease the stimulation of lipogenesis on refeeding.

Polymyxin B, a cyclic decapeptide antibiotic, increased blood glucose and lactate, and inhibited the stimulation of lipogenesis in interscapular brown adipose tissue and lactating mammary gland of starved-refed virgin and lactating rats respectively. Lipogenesis was not inhibited in white adipose tissue or liver. The antibiotic increased the haematocrit. The relative blood flow to brown adipose tissue and lactating mammary gland was decreased by polymyxin B, and this was accompanied by a decrease in tissue ATP content. In vitro polymyxin B did not affect glucose utilization or conversion into lipid, nor the stimulation by insulin of these processes in brown-adipose-tissue slices. Treatment of rats in vivo with polymyxin B resulted in decreased utilization of glucose in vitro in brown-adipose-tissue slices. Similarly, acini from mammary glands of polymyxin B-treated lactating rats had decreased rates of conversion of [1-14C]glucose to lipid. It is concluded that the effects of polymyxin B may be brought about by decreases in tissue blood flow. The possibility that these effects are secondary to inhibition of glucose utilization cannot be ruled out.     

Effect of chloramphenicol on ribonucleic acid synthesis in liver cells in suspension

1. Chloramphenicol has a stimulatory effect on the incorporation of radioactive phosphate into the RNA of perfused rat-liver slices, whole liver homogenates or the liver-cell suspensions, and no effect on the incorporation of [(14)C]adenine and [(14)C]uracil into the RNA of the tissue slices. 2. Chloramphenicol completely inhibits the incorporation of labelled adenine and uracil into the RNA of the cell suspensions, or into the RNA of homogenates derived from the whole liver tissues. 3. Chloramphenicol has at most a slight inhibitory effect on the transport of labelled adenine or uracil in the hepatic cells in suspension; in the slices, the transport of these bases is not inhibited at all. 4. The above observations indicate that: (a) unlike the tissue slices, hepatic cells in suspension are permeable to chloramphenicol; (b) in the presence of chloramphenicol, for reasons that are not clear, the conversion of the base into the appropriate nucleotide does not proceed.     

Metabolism of tissue cells in suspension. Synthesis of ribonucleic acid by liver cells in suspension

1. Liver cells in suspension are shown to incorporate several RNA precursors into their RNA. 2. The incorporation of [³²P]phosphate and [¹⁴C]adenine into the RNA of the cell suspension is usually of the same order as that in the perfused (or unperfused) liver slices. However, the initial lag in the incorporation of adenine into the RNA of the cell suspensions is much longer than that obtained for the tissue slices, and the optimum incorporation of adenine in the former, unlike that in the latter, needs exogenous glucose and probably a high concentration of phosphate. 3. The cell suspensions also differ from the tissue slices in being unable to incorporate [¹⁴C]orotic acid into their RNA, and resemble tumour tissues in incorporating uracil into their RNA at a rate significantly higher than that obtained with the tissue slices. 4. The above differences in the metabolic behaviour of liver-cell suspensions and tissue slices are considered to be due to the different levels of organization of the liver cells in the two tissue preparations.     

Methylation of newly synthesized ribonucleic acid by isolated rat liver nuclei. Characterization of the ribonucleic acid synthesized by nuclei from starved animals

1. Nuclei from rat liver incubated with S-adenosyl[methyl-(14)C]methionine incorporated radioactivity into RNA and into lipid and protein. 2. All of the labelled RNA was extracted from the nuclei with trichloroacetic acid at 90 degrees C. 3. The [(14)C]methyl-group incorporation into the hot-trichloroacetic acid extract was 30% inhibited by the addition of actinomycin D (100mug/mg of DNA) or by the omission of CTP, GTP and UTP. 4. Assuming that the main substrate for this triphosphate-dependent methylation was newly synthesized precursor rRNA containing one methyl group/30 uridylate residues, it was calculated that approx. 60% of the [(14)C]UMP incorporated under similar conditions represented precursor rRNA synthesis. 5. In agreement with this, low concentrations of actinomycin D (approx. 1mug/mg of DNA) sufficient to abolish the triphosphate-dependent incorporation of [(14)C]methyl group inhibited 68% of the [(14)C]UMP incorporation. 6. The incorporation of [(14)C]UMP by nuclei from starved animals decreased progressively with increasing periods of starvation, whereas the triphosphate-dependent [(14)C]methyl-group incorporation was not further decreased after 1 day of starvation. 7. This suggests that precursor rRNA synthesis decreased within 1 day whereas other species of RNA were affected only after longer periods of starvation.     

Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro.

1. The turnover rate of L-[1-14C]leucine was increased by 35% in lactating rats compared with virgin rats. Starvation or removal of pups (24 h) returned the value to that of the virgin rat. 2. Incorporation of L-[U-14C]leucine into lipid and protein of mammary glands of lactating rats in vivo increased 7-fold and 6-fold respectively compared with glands of virgin rats. Lactation caused no change in the incorporation of L-[U-14C]leucine into hepatic lipid and protein. 3. The production of 14CO2 from L[l-14C]leucine (in the presence of glucose) was similar in isolated acini from glands of fed (chow) and starved lactating rats. Feeding with a 'cafeteria' diet caused a slight decrease, and removal of pups a large decrease, in the oxidative decarboxylation of leucine. 4. Oxidation of L-[2-14C]leucine to 14CO2 was increased about 3-fold in acini from starved lactating rats or lactating rats fed on a 'cafeteria' diet compared with rats fed on a chow diet. Insulin decreased the formation of 14CO2 in all three situations. 5. Incorporation of L-[U-14C]- and [2-14C]-leucine into lipid was decreased in acini from starved lactating rats and lactating rats fed on a 'cafeteria' diet. Insulin tended to increase the conversion of [2-14C]leucine into lipid, but this was significant only in the case of the acini from 'cafeteria'-fed rats. 6. Experiments with (-)-hydroxycitrate indicate that the major route for conversion of leucine carbon into lipid in acini is via citrate translocation from the mitochondria. 7. The physiological implications of these findings are discussed.     

Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands.

Epidermal growth factor (EGF) is known to stimulate mammary epithelial proliferation, has been identified in milk and is expressed in lactating mammary epithelia. This study examined hormonal control of EGF mRNA in mammary glands of mice. Prepro-EGF mRNA (4.7 kb) was detected during lactation (and increased significantly during this period), whereas a smaller EGF-like RNA (.5 kb) was at highest levels in mammary glands of virgin and pregnant mice. The 4.7 kb RNA was polyadenylated, whereas .5 kb RNA was not. In mammary gland organ cultures from steroid-primed mice, the combinations of insulin + hydrocortisone and insulin + prolactin + hydrocortisone increased both prepro-EGF and beta-casein mRNA expression. When hydrocortisone was present there was a decrease in mammary gland content of EGF-like RNA (.5 kb band). We conclude that prepro-EGF mRNA expression in mouse mammary tissue is under the control of the lactogenic hormones prolactin and hydrocortisone.     

Inhibition by low concentrations of ouabain of prolactin-induced lactogenesis in rabbit mammary-gland explants

1. Explants of mammary tissue from pseudopregnant rabbits were cultured at 37 degrees C in air for 24-48h in Medium 199 buffered with 20mm-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The medium contained insulin and corticosterone, or insulin, corticosterone and sheep prolactin in the presence or absence of ouabain, an inhibitor of Na(+)/K(+)-dependent adenosine triphosphatase. The responses of explants were assessed histologically, by measuring the tissue concentration of K(+), and by rates of synthesis of RNA, protein and fatty acids. The effect of ouabain on Na(+) and K(+) concentrations in slices of lactating rabbit mammary-gland tissue incubated for 1h at 37 degrees C in Krebs bicarbonate buffer was also studied. 2. Prolactin increased the concentration of K(+) in mammary explants, an effect prevented by ouabain. In slices of lactating tissue, there was a linear relationship between the log dose of ouabain (from 0.1 to 10mum) and increased Na(+) and decreased K(+) concentrations in the tissue. 3. Ouabain at concentrations up to 1mum did not affect the rate of synthesis of RNA, protein or fatty acids by explants cultured with insulin and corticosterone. By contrast, the stimulatory effect of prolactin on protein synthesis was diminished and the induction of medium-chain fatty acid synthesis by prolactin was almost abolished. RNA synthesis was unaffected. Histological examination showed no tissue damage by 1mum-ouabain. 4. Explants cultured in the presence of 2mum-ouabain for 24h retained their ability to respond to prolactin when the ouabain was removed from the culture medium. Between 24 and 48h they showed responses to prolactin of a magnitude similar to those of explants never exposed to ouabain. 5. These results show that a fully functional Na(+)/K(+)-dependent adenosine triphosphatase system is necessary for prolactin to promote secretory activity in rabbit mammary gland.     

Tissue-specific effects of starvation and refeeding on the disposal of oral [1-14C]triolein in the rat during lactation and on removal of litter.

1. The effects of starvation and refeeding on the disposal of oral [14C]triolein between 14CO2 production and 14C-lipid accumulation in tissues of virgin rats, lactating rats and lactating rats with pups removed were studied. 2. Starvation (24 h) increased 14CO2 production in lactating rats and lactating rats with pups removed to values found in virgin rats. This increase was accompanied by decreases in 14C-lipid accumulation in mammary gland and pups of lactating rats and in white and brown adipose tissue of lactating rats with pups removed. 3. Short-term (2 h) refeeding ad libitum decreased 14CO2 production in lactating rats and lactating rats with pups removed, and restored the 14C-lipid accumulation in mammary glands plus pups and in white and brown adipose tissue respectively 4. Insulin deficiency induced with mannoheptulose inhibited the restoration of 14C-lipid accumulation in white adipose tissue on refeeding of lactating rats with pups removed, but did not prevent the restoration of 14C-lipid accumulation in mammary gland. 5. Changes in the activity of lipoprotein lipase in mammary gland and white adipose tissue paralleled the changes in 14C-lipid accumulation in these tissues. 6. It is concluded that 14C-lipid accumulation in mammary gland may not be affected by changes in plasma insulin concentration and that it is less sensitive to starvation than is lipogenesis or lactose synthesis. This has the advantage that the milk lipid content can still be maintained from hepatic very-low-density lipoprotein for a period after withdrawal of food. The major determinant of the disposal of oral 14C-triolein appears to be the total tissue activity of lipoprotein lipase. When this is high in mammary gland (fed lactating rats) or white adipose tissue (fed lactating rats with pups removed), less triacylglycerol is available for the muscle mass and consequently less is oxidized.     

The effect of thyroidectomy on the pattern of fatty acids synthesized by mammary gland from lactating rats

Thyroidectomy decreased the content of short-chain fatty acids and increased the content of long-chain fatty acids in the mammary glands of lactating rats. This effect was replicated in the glands of untreated rats limited to the same food intake as the thyroidectomized animals. Thyroidectomy decreased the incorporation of [6-(14)C]glucose into short-chain fatty acids and increased the incorporation into longer-chain acids. Restriction of the food intake of untreated animals did not cause a similar shift of the incorporation pattern. The possibility that the thyroxine effect on lipogenesis is secondary to its effect on carbohydrate metabolism is discussed.     

Effect of denervation on the synthesis of ribonucleic acid and deoxyribonucleic acid in rat diaphragm muscle

1. The effect of unilateral denervation of rat diaphragm muscle on its content of nucleic acids and their incorporation of precursors was investigated. 2. After denervation the paralysed hemidiaphragm hypertrophies and within 3 days its content of RNA increases considerably. The concentration of DNA/unit mass remains fairly constant. 3. During this period there is some increase in the rate of incorporation of [(14)C]adenine into RNA, whereas there is some diminution in the rate of incorporation of [(14)C]orotic acid. 4. Incorporation of [(14)C]adenine and [(3)H]thymidine into DNA is much increased in the paralysed tissue, reaching its maximum by about the third day, but returning to normal by the tenth. 5. The significance of these results in relation to the hypertrophy after denervation is discussed.     

Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate.

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.     

Stimulation of incorporation of nucleic acid precursors into HeLa cells caused by provaline

1. The effect of proflavine and other acridines on the incorporation of precursors into the nucleic acids of HeLa cells was examined. 2. Relatively low concentrations (50mum) of proflavine completely inhibited incorporation of precursors into DNA, but allowed a small extent of incorporation into RNA. 3. Acridine-resistant incorporation into RNA was unaffected by actinomycin D at 2mug./ml. and persisted even at high concentrations (500mum) of many acridines. 4. A few combinations of acridine and precursor, notably 250mum-proflavine and [(14)C]adenine, caused a stimulation of incorporation. 5. The proflavine-stimulated incorporation was into alkali-stable di- and tri-nucleotides. 6. It was concluded that the effect was due to the preferential inhibition of degradation of a fraction of RNA that normally turned over, thus allowing small radioactive oligonucleotides to accumulate in the cells.     

Polyphasic changes in incorporation of precursors into ribonucleic acid of oestradiol-stimulated mammary gland

1. At 3 weeks after ovariectomy, mammary glands (5th pair) of adult Swiss mice show (i) no significant decrease in weight, (ii) 20% of the original rate of incorporation of [(3)H]-uridine into RNA (after a 30min pulse), and (iii) 90% of the original rate of incorporation of l-[(3)H]leucine into protein (after a 15min pulse). 2. A single injection of oestradiol-17beta into these ovariectomized mice produces, during the next 17h, a series of discrete bursts of increased incorporation of [(3)H]uridine into mammary-gland RNA; the bursts, which are variable in height, reach peaks at approx. 1, 9, 12 and 16h after hormone administration; an increase is already detected at 15min, the earliest time-point investigated; each burst lasts for approx. 2h. There is no significant stimulation of [(3)H]uridine incorporation into RNA of liver and quadriceps femoris muscle. 3. Nuclear incorporation of [(3)H]UTP into RNA of mammary gland in vitro is linear with time for up to 20min at 15 degrees C; it requires CTP, GTP and ATP and is inhibited by actinomycin D. Also, the incorporation is strongly inhibited by alpha-amanitin in high salt concentrations but only weakly in low salt concentrations, a result indicating that RNA polymerase II activity predominates in high salt, whereas RNA polymerase I activity predominates in low salt concentrations. Injection of oestradiol-17beta in vivo followed by measurement of nuclear RNA synthesis in vitro shows a definite increase in both RNA polymerase activities 30min after oestradiol-17beta injection, the earliest time-point investigated, a higher increase at 1h, a decline at 4h, and again a large increase at 12h. These results in general agree with the changes in precursor incorporation into RNA measured directly in the animal and suggest that changes in [(3)H]uridine uptake into RNA are not precursor-pool-dependent.     

Chylomicron margination, lipolysis, and vitamin a uptake in the lactating rat mammary gland: implications for milk retinoid content

We have reported previously that the concentration of vitamin A (VA) in the milk of lactating rats varies with dietary VA intake, even when plasma retinol concentration is unaffected. In the current study, we investigated the role of lipolysis in the uptake of chylomicron (CM) VA into mammary tissue of lactating rats and estimated the proportion of CM-VA that is associated with the mammary gland during CM clearance. Chylomicrons containing [(3)H]VA, mainly as retinyl esters, were prepared in donor rats and administered intravenously to lactating recipient rats. Chylomicron VA rapidly disappeared from plasma and appeared in mammary tissue (maximum within 2-3 mins), followed by a decline. Concomitantly, uptake by liver increased continuously, reaching a plateau within 20-30 mins. Active lipolysis in mammary tissue was necessary for rapid VA uptake, as significantly less CM-VA was recovered in mammary tissue of postlactating rats than of lactating rats, after heparin treatment in lactating rats, or after injection of preformed CM remnants in lactating rats. [(3)H]Vitamin A uptake by mammary tissue increased linearly with CM-VA dose over a 150-fold dose range (R(2) = 0.972, P = 0.0001), suggesting a high capacity for uptake and apparent first-order assimilation of CM-VA during CM remnant formation in situ. Model-based compartmental analysis using WinSAAM predicted that approximately 42% of CM-VA marginated, that is, were temporarily removed, from plasma to the mammary glands during lipolysis and that a total of 3.8% of CM-VA was transferred to mammary tissue. The model-predicted t(1/2) for CM remnants was 3.04 mins. The metabolism of CM-VA in the lactating mammary gland, in proportion to VA absorption and CM-VA contents, may explain how milk VA concentration varies even when plasma retinol levels are unchanged. The mechanism of CM margination and mammary gland uptake described here for VA may be similar for other lipophilic substances.     

Re-examination of the putative roles of insulin and prolactin in the regulation of lipid deposition and lipogenesis in vivo in mammary gland and white and brown adipose tissue of lactating rats and litter-removed rats.

1. The effects of various treatments to alter either plasma prolactin (bromocryptine administration or removal of litter) or the metabolic activity of the mammary gland (unilateral or complete teat sealing) on the disposal of oral [14C]lipid between 14CO2 production and [14C]lipid accumulation in tissues of lactating rats were studied. In addition, the rates of lipogenesis in vivo were measured in mammary gland, brown and white adipose tissue and liver. 2. Bromocryptine administration lowered plasma prolactin, but did not alter [14C]lipid accumulation in mammary gland or in white and brown adipose tissue. 3. In contrast, complete sealing of teats results in no change in plasma prolactin, but a 90% decrease in [14C]lipid accumulation in mammary gland and a 4-fold increase in white and brown adipose tissue. The rate of lipogenesis in mammary gland was decreased by 95%, but there was no change in the rate in white and brown adipose tissue. Unilateral sealing of teats resulted in a decrease in [14C]lipid accumulation in white adipose tissue. 4. Removal of the litter for 24 h (low prolactin) produced a similar pattern to complete teat sealing, except that there was a 6-fold increase in lipogenesis in white adipose tissue. Re-suckling for 5 h increased plasma prolactin, but did not alter the response seen in litter-removed lactating rats. 5. Changes in lipoprotein lipase activity and in plasma insulin paralleled the reciprocal changes in [14C]lipid accumulation in white and brown adipose tissue and in mammary gland. 6. It is concluded that the plasma insulin is more important than prolactin in regulating lipid deposition in adipose tissue during lactation, and that any effects of prolactin must be indirect.     

Effects of pancreozymin, urecholine and actinomycin D on the metabolism of ribonucleic acid by canine pancreas

1. Canine pancreas slices were incubated with [6-(14)C]orotic acid and the rate of its incorporation into RNA was measured. RNA was fractionated by shaking homogenates with phenol at 2 degrees , 50 degrees , 65 degrees and 80 degrees . Cytoplasmic RNA was extracted at the lowest temperature and nuclear RNA at the higher temperatures. The samples were centrifuged through sucrose gradients and the E(260) and (14)C-sedimentation patterns determined. Incorporation of orotic acid was very rapid into cytoplasmic 4s RNA. This probably represents end-group turnover. No incorporation into cytoplasmic ribosomal RNA was observed. 2. The nuclear 50 degrees -RNA exhibited two E(260) peaks, at 18s and 28s. This portion of the sample contained but moderate amounts of [(14)C]RNA. The highly labelled material had sedimentation coefficients in the range 35-50s. The nuclear 65 degrees -RNA showed an E(260) peak at 16s. The [(14)C]RNA peak occurred at 25-35s and this portion demonstrated the highest specific activity of any RNA fraction. 3. The 50 degrees -RNA, 65 degrees -RNA and 80 degrees -RNA were hydrolysed and their base compositions were determined. All three samples possess a ribosomal type of composition (G+C)/(A+U)=(1.4-1.7). For this reason they are considered to contain ribosomal precursor RNA as their major constituent. 4. Actinomycin D (0.5mug./ml.) in the incubation medium inhibited incorporation of orotic acid into both nuclear fractions but not into 4s RNA. 5. The cholinergic drug Urecholine inhibited incorporation into the heavy, high-specific-activity portions of the nuclear fractions but did not inhibit incorporation into the ribosomal precursor type of nuclear RNA. A similar result was also obtained with the hormone pancreozymin. Moderate inhibition of incorporation of orotic acid into 4s RNA likewise resulted from the presence of the drug and the hormone.     

Interactions of glucose, acetoacetate and insulin in mammary-gland slices of lactating rats.

1. Utilization of 5mM-glucose by slices of lactating mammary gland was decreased 33% on addition of acetoacetate (2mM) to the incubation medium. This inhibition was accompanied by increases in the intracellular concentrations of citrate and glucose 6-phosphate. 2. In the presence of acetoacetates the accumulation of pyruvate in the medium approximately doubled. 3. Insulin completely reversed the inhibitory effect of acetoacetates on glucose utilization, without altering the amount of acetoacetate removed or pyruvate formed. 4. Similar results were obtained with mammary-gland slices from diabetic rats, except that insulin did not completely reverse the effects of acetoacetates. 5. Acetoacetate inhibited the formation of 14CO2 from [1-14C]pyruvate; this effect was not overcome by insulin. 6. Insulin increased the proportion of [3-14C]acetoacetate that was converted into lipid and decreased that oxidized to CO2.7. The physiological significance of these findings is discussed.