AMINO ACID DISTRIBUTION AND INCORPORATION INTO PROTEINS IN ISOLATED, ELECTRICALLY-STIMULATED CEREBRAL TISSUES

—The uptake of radioactive amino acid by incubated cerebral cortex slices is found to be a first order process. Incorporation of the radioactive amino acid into tissue protein is from a precursor pool that has first equilibrated with the intracellular endogenous free amino acids. Ways of calculating the amino acid incorporation in molar quantities from the observed incorporation of radioactivity are discussed, and it is concluded that the specific radioactivity of the intracellular acid-soluble fraction is the best basis for such estimates. The in vitro incorporation of leucine into tissue protein is estimated to be approximately 1±2 mμnol/mg protein/h, and of valine 0±4 mμmol/mg protein/h. Addition of free amino acids to the media had little or no effect on the calculated rates of incorporation. On incubation for 1 h the total free valine in tissue and medium increased by 0±43 μmol/g and leucine increased by 0±55 μmol/g. Estimates of amino acid incorporation based on the specific radioactivity of the media amino acids can give misleading results if this considerable release of amino acids into the medium is not taken into account. Electrical stimulation of neocortical slices with a variety of types of pulses was either without effect or decreased incorporation into portein. The decrease could not be directly correlated with changes in tissue K⁺ nor with the utilization of ATP. Mild, local stimulation of the lateral olfactory tract of piriform cortex slices was without effect on tissue phosphocreatine, K⁺ or amino acid incorporation.     

The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat.

The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per mug DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. The absence of a short term effect of insulin on the rate of protein synthesis does not exclude a long term effect as suggested by the higher rate of incorporation in the cells of peri-insular acini.     

The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo

1. Rats were infused in vivo with [U-(14)C]glycine for periods of 2-6h, during which time the specific radioactivity of the free glycine in plasma and tissue approached a constant value. 2. Free serine also became labelled. The ratio of specific radioactivity of serine to that of glycine in the protein of liver, kidney, brain, jejunum, heart, diaphragm and gastrocnemius muscle was closer to the ratio in the free amino acid pool of the tissue than that of the plasma. 3. The kinetics of incorporation of [(14)C]glycine and [(14)C]serine into the protein of gastrocnemius muscle further suggested that the plasma free amino acids were not the immediate precursors of protein. 4. Infusion of rats with [U-(14)C]serine resulted in labelling of free glycine. The ratio of specific radioactivity of glycine to serine in the protein of liver, kidney, brain, jejunum and heart again suggested incorporation from a pool similar to the free amino acid pool of the tissue. 5. Rates of tissue protein synthesis calculated from the incorporation into protein of both radioactive glycine and serine, either infused or derived, were very similar when the precursor specific radioactivity was taken to be that in the total free amino acids of the tissue. Except for gastrocnemius muscle and diaphragm during the infusion of radioactive serine, the rates of tissue protein synthesis calculated from the specific radioactivity of the free glycine and serine in plasma differed markedly.     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : III. Tubular Gland Cell Cytodifferentiation

Administration of estrogen (E) to immature chicks triggers the cytodifferentiation of tubular gland cells in the magnum portion of the oviduct epithelium; these cells synthesize the major egg-white protein, ovalbumin. Electron microscopy and immunoprecipitation of ovalbumin from oviduct explants labeled with radioactive amino acids in tissue culture were used to follow and measure the degree of tubular gland cell cytodifferentiation. Ovalbumin is undetectable in the unstimulated chick oviduct and in oviducts of chicks treated with progesterone (P) for up to 5 days. Ovalbumin synthesis is first detected 24 hr after E administration, and by 5 days it accounts for 35% of the soluble protein being synthesized. Tubular gland cells begin to synthesize ovalbumin before gland formation which commences after 36 hr of E treatment. When E + P are administered together there is initially a synergistic effect on ovalbumin synthesis, however, after 2 days ovalbumin synthesis slows and by 5 days there is only 1/20th as much ovalbumin per magnum as in the E-treated controls. Whereas the magnum wet weight doubles about every 21 hr with E alone, growth stops after 3 days of E + P treatment. Histological and ultrastructural observations show that the partially differentiated tubular gland cells resulting from E + P treatment never invade the stroma and form definitive glands, as they would with E alone. Instead, these cells appear to transform into other cell types—some with cilia and some with unusual flocculent granules. We present a model of tubular gland cell cytodifferentiation and suggest that a distinct protodifferentiated stage exists. P appears to interfere with the normal transition from the protodifferentiated state to the mature tubular gland cell.     

Effect of gonadal steroids and gamma-aminobutyric acid on LH release and dopamine expression and activity in the zona incerta in rats

A dopaminergic system in the zona incerta stimulates LH release and may mediate the positive feedback effects of the gonadal steroids on LH release. In this study the mechanisms by which steroids might increase dopamine activity in the zona incerta were investigated. In addition, experiments were conducted to determine whether the inhibitory effects of gamma-aminobutyric acid (GABA) on LH release in the zona incerta are due to suppression of dopamine activity in this area or conversely whether the stimulatory effects of dopamine on LH release are due to suppression of a tonic inhibitory GABAergic system. Ovariectomized rats were treated s.c. with oil, 5 micrograms oestradiol benzoate or 5 micrograms oestradiol benzoate followed 48 h later by 0.5 mg progesterone, and killed 54 h after the oestradiol benzoate injection. At this time the LH concentrations were suppressed in the oestradiol benzoate group and increased in the group treated with oestradiol benzoate and progesterone. The ratio of tyrosine hydroxylase:beta-actin mRNA in the zona incerta was significantly increased by the oestradiol benzoate treatment, but the addition of progesterone resulted in values similar to those in the control group. At the same time, the progesterone treatment increased tyrosine hydroxylase activity in the zona incerta as indicated by an increase in L-dihydroxyphenylalanine (L-DOPA) accumulation after 100 mg 3-hydroxybenzylhydrazine hydrochloric acid (NSD1015) kg-1 and an increase in dopamine release as indicated by a increase in dihydroxyphenylacetic acid (DOPAC) concentrations (one of the major metabolites of dopamine). Ovariectomized rats treated with oestradiol benzoate plus progesterone were also injected i.p. with 75 mg gamma-acetylenic GABA kg-1 (a GABA transaminase inhibitor) to increase GABA concentrations in the brain. This treatment had no effect on the ratio of tyrosine hydroxylase:beta-actin mRNA but decreased L-DOPA accumulation and DOPAC concentrations in the zona incerta, indicating a post-translational inhibition of dopamine synthesis and release. Treatment of ovariectomized rats with oestradiol benzoate followed by 100 mg L-DOPA i.p. to increase dopamine concentrations in the whole brain had no effect on glutamic acid decarboxylase mRNA expression in the zona incerta, although it increased the glutamic acid decarboxylase:beta-actin mRNA ratio in other hypothalamic areas (that is, the medical preoptic area, ventromedial nucleus and arcuate nucleus). In conclusion, the steroids act to increase dopamine activity in different ways: oestrogen increases tyrosine hydroxylase mRNA expression and progesterone acts after translation to increase tyrosine hydroxylase activity and dopamine release (as indicated by increases in DOPAC concentrations). This latter effect may be due to progesterone removing a tonic GABAergic inhibition from the dopaminergic system.     

Regulation of avidin accumulation by prostaglandins in chick oviduct culture

Regulation of avidin accumulation by prostaglandins (PGs) and their inhibitors was studied in chick oviduct organ culture. Avidin was induced neither by progesterone nor PGF2 alpha in the oviduct of immature chicks. By progesterone and PGs, a high avidin synthesis was induced when the chicks received diethylstilbestrol (DES) for 7 days. Enhanced avidin production was observed by PGF2 alpha, PGE1 and PGE2, whereas PGA2 and PGB2 had a slight inhibitory effect and PGA1 and PGB1 had no effect on avidin production. PGF2 alpha was most effective at a concentration of 10-20 micrograms/ml. The effects of progesterone and PGF2 alpha were not additive. Mefenamic acid, at concentrations of 40 and 60 micrograms/ml, inhibited 50 and 85%, respectively, of the avidin synthesis induced by progesterone, whereas the inhibition of the total protein synthesis was only 20%, and this only by the higher concentration of the drug. Tolfenamic and meclofenamic acid were also inhibitory in the case of progestin-induced avidin synthesis. These studies indicate that the PGs (F2 alpha, E1 and E2) might be involved in the avidin induction in the chick differentiated oviduct. The specific inhibition of the progesterone-dependent avidin synthesis by the PG inhibitors suggests that PGs may be connected with the progesterone action in the oviduct. We propose that the avidin synthesis by the chick oviduct might be considered as a model system for studying PG effects on the synthesis of a specific protein.     

INTERACTION OF ESTROGEN AND PROGESTERONE IN CHICK OVIDUCT DEVELOPMENT : I. Antagonistic Effect of Progesterone on Estrogen-Induced Proliferation and Differentiation of Tubular Gland Cells

Daily administration of estrogen to immature female chicks results in marked oviduct growth and appearance of characteristic tubular gland cells which contain lysozyme. Although a rapid increase in total DNA and RNA content begins within 24 hr, cell specific protein, lysozyme, is first detectable after 3 days of estrogen. Progesterone administered concomitantly with estrogen antagonizes the estrogen-induced tissue growth as well as appearance of tubular gland cells and their specific products, lysozyme and ovalbumin. When the initiation of progesterone administration is delayed for progressively longer periods (days) during estrogen treatment, proportionally greater growth occurs with more lysozyme and tubular gland cells after 5 days of total treatment. Progesterone does not inhibit the estrogen-stimulated increase in uptake of α-aminoisobutyric acid and water by oviduct occurring within 24 hr or the estrogen-induced increase in total lipid, phospholipid, and phosphoprotein content of serum. The above results of progesterone antagonism can best be explained by the hypothesis that progesterone inhibits the initial proliferation of cells which become tubular gland cells but does not antagonize the subsequent cytodifferentiation leading to the synthesis of lysozyme and ovalbumin once such cell proliferation has occurred.     

Effects of amino acids, ammonia and leupeptin on protein synthesis and degradation in isolated rat hepatocytes.

Protein synthesis in isolated rat hepatocytes, as measured by the incorporation of [14C]-valine at constant specific radioactivity, proceeded at a rate of 0.3-0.5%/h in an unsupplemented medium, i.e. only about one-tenth the rate of protein degradation (4%/h). Leupeptin, which inhibits lysosomal protein degradation (previously found to be 75% of the total degradation in hepatocytes), had no effect on protein synthesis, showing that endogenous protein degradation supplied amino acids in excess of the substrate requirements for protein synthesis. The inhibition of protein synthesis by NH4Cl (another inhibitor of lysosomal protein degradation) as well as the stimulation by a physiological amino acid mixture must therefore represent indirect effects, either on general energy metabolism, or on unknown regulatory processes.     

Oestrogen and progesterone receptors in chick oviduct chromatin after administration of oestradiol, progesterone or anti-oestrogen.

Oestrogen-primed and withdrawn chicks were injected with oestradiol benzoate, progesterone, and/or the anti-oestrogens tamoxifen and 4-hydroxytamoxifen. Oestrogen receptors were studied in oviduct chromatin solubilized by mild digestion of purified nuclei with micrococcal nuclease. After a single injection of oestradiol benzoate, ultracentrifugation on sucrose gradients of chromatin extracts labelled with [3H]-oestradiol showed two peaks of oestradiol binding sites, sedimenting at 13--14 S and 7--8 S. After repeated injections of oestradiol benzoate, the 13--14 S peak increased more than the 7--8 S peak. After injection of anti-oestrogen alone or together with oestradiol benzoate, no [3H]oestradiol-binding or 4-hydroxy[3H]tamoxifen-binding peaks were detected in the chromatin. Injection of progesterone also produced an increase of the 13--14 S and 7--8 S chromatin oestradiol receptor. Progesterone receptor could only by detected in chromatin early after progesterone administration, and it sedimented in density gradients with the 12 S mononucleosome fraction. Tamoxifen injected together with progesterone gave higher levels of 13--14 S oestrogen binding sites than did progesterone alone. The presence of a 13--14 S peak of oestrogen binding sites in hormonal situations which promote a biological response in the chick oviduct, and the absence of this peak after administration of anti-oestrogens, suggest that this subfraction of chromatin contains elements involved in gene regulation.     

Effect of estradiol and progesterone on oviductal LH-receptors and LH-dependent relaxation of the porcine oviduct

We have previously shown that the porcine oviduct possesses immunoreactive and functional LH receptors and that LH causes relaxation of the oviduct, especially during the periovulatory stage of estrous cycle. The current studies were undertaken to investigate the effects of estradiol and progesterone on LH receptor protein and LH-stimulated motility of the oviduct in steroid-primed ovariectomized gilts. Twenty-one cross-bred gilts were ovariectomized at 6 m.o. of age. Four weeks later gilts received daily intramuscular injection of either 2 mL corn oil (control n = 4), estradiol benzoate (EB) 1.5 mg (n = 6), progesterone 50 mg (n = 5), or 1.5 mg EB plus 50 mg progesterone (n = 6) for 4 consecutive days. The gilts were slaughtered on Day 5 after the first injection of steroids or vehicle. Rings of isthmus and ampulla were collected from each oviduct and placed in a tissue chamber perfused with Kreb's solution for 60 min. The mechanical activity was recorded for 30 min after LH treatment. Immunoreactivity of LHR in the Fallopian tube sections were detected in the epithelium of the tubal mucosa, smooth muscle cells and the blood vessel endothelium. Western blotting showed that porcine oviducts contain 75, 48 and 45 kDa immunoreactive LH receptor proteins, like the corpus luteum (CL). The lowest receptor expression was found in controls and in gilts treated with estradiol or progesterone. Combined treatment with estradiol and progesterone resulted in a significant increase of LH receptor protein concentrations when compared with control animals. In vitro LH treatment affected oviduct contractility of combined estradiol and progesterone treated gilts but not the oviduct of the remaining groups. It also caused a decrease in amplitude, frequency and areas under the curve (AUC) of ampulla (P < 0.05) and the amplitude and AUC of isthmus (P < 0.001) in combined estradiol and progesterone-primed gilts. These results indicate that estradiol and progesterone together, but not separately, increase LH receptor protein in the porcine oviduct and that combined estradiol and progesterone priming is necessary for LH-induced relaxation of the porcine oviduct.     

Effects of a luteolytic dose of oestradiol benzoate on uterine oxytocin receptor concentrations, phosphoinositide turnover and prostaglandin F-2 alpha secretion in sheep

Administration of oestradiol-17 beta benzoate on Days 9 and 10 of the oestrous cycle resulted in episodic secretion of PGF-2 alpha (as indicated by elevated circulating concentrations of 13,14-dihydro-15-ketoprostaglandin F-2 alpha) and a decline in circulating progesterone. Release of PGF-2 alpha began 35 +/- 3 h after first injection of oestrogen and progesterone concentrations declined from 42 +/- 3 h. Secretion of oxytocin, which was first observed 26 +/- 3 h after oestrogen treatment, preceded secretion of PGF-2 alpha; 69% of pulses of oxytocin coincided with episodes of PGF-2 alpha secretion. Uterine oxytocin receptor concentrations were raised in ewes treated with oestrogen, increases occurring in caruncular endometrium and myometrium by 12 h after treatment and in intercaruncular endometrium by 24 h. Raised receptor concentrations were followed at 24 h by increases in the incorporation of [3H]inositol into phosphatidylinositol and in the hydrolysis of labelled tissue phosphoinositides in response to oxytocin in slices of caruncular endometrium incubated in vitro. The following sequence of events is therefore suggested to occur at oestrogen-induced luteolysis: induction of the oxytocin receptor; increased turnover of phosphoinositides; onset of episodic secretion of PGF-2 alpha; and functional luteolysis.     

THE EFFECT OF MORPHINE ON THE TRANSPORT AND METABOLISM OF INTRACISTERNALLY-INJECTED LEUCINE IN THE RAT

—Intracisternally injected l or d-[¹⁴C]leucine was retained longer in the brains of morphine-treated rats than in saline-injected control animals. This resulted in higher levels of the labelled leucine and of labelled metabolites of the l-isomer in free pools of brain tissue. However, the absolute levels of brain amino acids and the relative distribution of radioactivity among l-leucine metabolites in brain were unaffected by treatment with morphine, indicating that no disturbance of leucine oxidation through the citric acid cycle was produced by the drug. The inhibition of protein synthesis caused by acute administration of morphine was calculated to be greater than previously reported since morphine treatment increased the specific radioactivity of the free pool of leucine in brain following the intracisternal injection of the labelled amino acid. Possible mechanisms responsible for these morphine effects are discussed.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

Monohydroxytamoxifen: an antioestrogen with high affinity for the chick oviduct oestrogen receptor

The monohydroxylated derivative of tamoxifen (a non-steroidal triaryl ethylene antioestrogen) shows an apparent affinity (Ki = 0.2 nM) for the chick oviduct oestrogen receptor which is higher than that of oestradiol itself, and ～ 10 times higher than that of tamoxifen. Administered $\text{in}\text{vivo}$ with oestradiol benzoate, it inhibited the increase of tissue growth, progesterone receptor content, ornithine decarboxylase activity (ODC), and ovalbumin and conalbumin synthesis, and also inhibited the oestradiol induced increase of ODC $\text{in}\text{vitro}$. It did not display any oestrogenic effect by itself. We conclude that antioestrogenic action may be exhibited by a molecule with higher affinity binding to the oestrogen receptor than oestradiol itself. Metabolic studies demonstrated that the antioestrogenic action of tamoxifen is not due to its prior conversion to monohydroxytamoxifen.     

Amino acid incorporation by cell fractions from the oviduct of the laying hen and the synthesis of egg-white proteins

1. Homogenates of the magnum of the hen oviduct have been fractionated by differential centrifuging. 2. Centrifuging at 600g for 10min. gave a pellet containing most of the particulate material of the cell, but on washing this fraction some particles were removed from it. The washed 600g pellet contained most of the DNA of the homogenate. 3. Centrifuging the 600g supernatants at 10000g for 10min. gave particulate fractions (I particles) richer in RNA than other fractions which were active in incorporating amino acids into protein in isolation. When minced tissue was incubated with radioactive amino acids before homogenizing these particles were the most radioactive of the cell fractions. 4. The pellet obtained by centrifuging the 10000g supernatant at 105000g for 60min. was very small; its RNA/protein ratio was raised compared with that of the homogenate. It only incorporated amino acids in isolation to a small extent or not at all. 5. The 105000g supernatant contained a large proportion of the protein of the homogenate. 6. The I particles could be subfractionated by layering over denser sucrose to give a pellet with lower RNA content and incorporating activity, and also suspended material richer in both these properties. 7. Treatment of the I particles with deoxycholate gave rise to particles sedimenting at 105000g for 60min. containing most of the RNA of the original particles, but only about 34% of the protein, and with a high activity in incorporating amino acids. 8. The I particles, or particles derived from them by deoxycholate treatment, required GTP and phosphoenolpyruvate for the incorporation of free amino acids. The omission of ATP reduced the incorporation to varying extents. Chicken-liver cell sap stimulated the activity. 9. Radioactive amino acids linked to transfer RNA were transferred to protein by I particles. GTP and phosphoenolpyruvate were required for this transfer. The phosphoenolpyruvate requirement could not be replaced by increasing the GTP concentration. ATP partially inhibited the transfer. 10. After incorporation by the cell-free system, the hot-trichloroacetic acid extract, but not the lipid extract, was radioactive. Ribonuclease and puromycin inhibited at low concentrations. Lecithinase-C was much less inhibitory. Transfer of amino acid, from a radioactive lipid-amino acid complex prepared from hen oviduct, was not detected. 11. After short periods of incubation of minced tissue with [(14)C]lysine some of the radioactive protein of the isolated I particles behaved as ovalbumin. The distribution of radioactivity in the protein resembled that in ovalbumin in soluble extracts of the tissue obtained after longer periods of incubation.     

Effects of human chorionic gonadotrophin on contractile activity of steroid-primed pig myometrium in vitro

We examined the effects of (a) oestrogen and progesterone on concentrations of luteinizing hormone/human chorionic gonadotrophin (LH/hCG) receptors in uterine smooth muscle in vivo and (b) hCG on spontaneous myometrial contractions in vitro. Ovariectomized gilts received 2 ml corn oil (control; n = 5), 2 mg oestradiol benzoate (n = 6) or 20 mg progesterone (n = 5) for 5 days. Gilts were hysterectomized 8 h after the last injection and longitudinal sections of myometrium were incubated in modified Krebs' solution with 0 or 10 i.u. of hCG (n = 10/gilt) for 4 h at 37 degrees C in 95% O2:5% CO2. After incubation, myometrial sections were placed in a tissue chamber perfused with Krebs' solution and mechanical activity was recorded for 30 min. Cell membrane fractions were prepared from myometrial tissue not used for in-vitro studies and analysed for LH/hCG receptors. Treatment with oestradiol benzoate increased (P less than 0.01) the number of LH/hCG-binding sites compared with gilts receiving corn oil or progesterone. Incubation of myometrium with hCG reduced (P less than 0.01) the frequency and amplitude of spontaneous uterine contractions in gilts treated with oestradiol benzoate. In contrast, hCG had no effect (P greater than 0.05) on the pattern of myometrial contractions in gilts given corn oil or progesterone. These results indicate that oestradiol promotes the synthesis of LH/hCG receptors in pig myometrium and incubation of oestrogen-primed tissue with hCG has a quiescent effect on myometrial contractility.     

Intracellular amino acids and protein synthesis in Hela cells

1. When the intracellular amino acid pool is prelabelled and subsequently chased in non-radioactive medium, the radioactivity of the amino acid pool is not found to have been incorporated into protein.
2. Leucine transport into Hela cells is reduced in the presence of 10 mM valine in the medium. This results in a lower specific radioactivity of leucine in the intracellular amino acid pool. However, neither the overall rate of protein synthesis nor the incorporation of radioactive leucine into protein is affected.

From these experiments it is concluded that incoming amino acids entering the intracellular amino acid pool are not used for de novo synthesis of protein.     

Acceptor sites for the oestrogen receptor in hen oviduct chromatin.

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.