Control of growth hormone production by a clonal strain of rat pituitary cells. Stimulation by hydrocortisone

Addition of hydrocortisone to the medium of a clonal strain of rat pituitary cells (GH₃) stimulated the rate of production of growth hormone. The stimulation had a lag period of about 24 hr, reached a maximum at 70–100 hr, and was observed at a hydrocortisone concentration as low as 5 x 10^-8 M. Cells maximally stimulated with 3 x 10^-6 M hydrocortisone produced 50–160 µg growth hormone/mg cell protein/24 hr. These rates were four to eight times those observed in control cells. At maximum stimulation, intracellular levels of growth hormone in both stimulated and control cells were equal to the amount secreted into the medium in about 15 min. Removal of hydrocortisone from the medium of GH³ cells caused a return of the rate of growth hormone production to that in control cells. Addition of hydrocortisone to the medium of cells growing exponentially with a population-doubling time of 60 hr caused both an increase in the doubling time to 90 hr and a stimulation of growth hormone production. Cycloheximide (3.6 x 10^-5 M) and puromycin (3.7 x 10^-4 M) suppressed incorporation of labeled amino acids into protein by 93 and 98%, respectively, and suppressed growth hormone production by stimulated and control cells by at least 94%.     

Effect of osmolality on the hydraulic permeability coefficient of red cells

The osmotic water permeability coefficient, L_p, for human and dog red cells has been measured as a function of medium osmolality, and found to depend on the osmolality of the bathing medium. In the case of human red cells L_p falls from 1.87 x 10^-11 cm³/dyne sec at 199 mOSM to 0.76 x 10^-11 cm³/dyne sec at 516 mOSM. A similar decrease was observed for dog red cells. Moreover, L_p was independent of the direction of water movement and the nature of the solute used to provide the osmotic pressure gradient; it depended only on the final osmolality of the medium. Furthermore, L_p was not affected by pH in the range of 6 to 8 nor by the presence of drugs such as valinomycin (1 x 10^-6 M) and tetrodotoxin (3.2 x 10^-6 M). The instantaneous nature of the response to changes in external osmolality suggests that the hydraulic conductivity of the membrane is controlled by a thin layer at the outer face of the membrane.     

Induced changes in the rates of uridine- 3 H uptake and incorporation during the G 1 and S periods of synchronized Chinese hamster cells

The rates of uridine-5-³H incorporation into RNA and the rates of uridine uptake into the acid-soluble pool during the cell cycle of V79 Chinese hamster cells were examined. Cells cultured on Eagle''s minimal essential medium supplemented with fetal calf serum, lactalbumin hydrolysate, glutamine, and trypsin displayed rates of incorporation and uptake which increased only slightly during G₁ and accelerated sharply as DNA synthesis commenced. In contrast, cells cultured on minimal essential medium supplemented only with calf serum exhibited rates of incorporation and uptake which increased linearly through both G₁ and S. The transition from one pattern to the other can be induced within 24 hr and is completely reversible. The nonlinear pattern exhibited by cells grown on the supplemented fetal calf serum medium can also be overcome with high exogenous uridine concentrations. In the presence of 200 µM uridine, these cells display a linear pattern of increase in rates of uridine incorporation and uptake. It is concluded that at lower uridine concentrations the pattern of increase in the rate of uridine incorporation into RNA during the cell cycle for a given population of cells is dependent upon the rate of uridine entry into the cell, and that this pattern is not rigidly determined but can be modified by culture conditions.     

Structure-activity relationships of several cardiotonic steroids with respect to inhibition of ion transport in frog muscle

Cesium uptake by sodium-loaded frog sartorius muscles was inhibited 100% by 10^-6 M ouabain and 10^-6 M cymarin. The doses for 50% inhibition of cesium uptake by five cardiotonic aglycones were 1.5 x 10^-6 M for strophanthidin, 2 x 10^-7 M for telocinobufagin, 1.6 x 10^-6 for digitoxigenin, 2.4 x 10^-6 M for periplogenin, and 6.3 x 10^-6 M for uzarigenin. Because of the limited solubility of sarmentogenin the maximum concentration studied was 2 x 10^-6 M which inhibited cesium uptake about 36%. Inhibition of cesium uptake by cymarin was not reversed during a 3.5 hr incubation in fresh solution while the muscles treated with ouabain and strophanthidin recovered partly during this time. Cymarin was a more potent inhibitor of sodium efflux than strophanthidin and periplogenin was less potent. Increased cesium ion concentration in the external solution decreased the strophanthidin inhibition of cesium uptake but 25 mM cesium did not overcome the inhibition by 10^-8-10^-6 M strophanthidin. Increased potassium ion concentration in the external solution decreased but did not completely overcome inhibition of sodium efflux by strophanthidin. It is concluded that potassium or cesium ions do not compete with these drugs for a particular site on the ion transport complex. The same structural features of the drugs are necessary for inhibition of ion transport in frog muscle as are required for inhibition of ion transport in other tissues, inhibition of sodium-potassium-stimulated ATPases, and toxicity to animals.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : I. Production of Stable, Reversible G1-Arrested Populations in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO) were grown to stationary phase (approximately 8–9 x 10⁵ cells/ml) in F-10 medium. Cells remained viable (95%) for at least 80 hr in stationary phase, and essentially all of the cells were in G₁ Upon resuspension or dilution with fresh medium, the cells were induced to resume traverse of the life cycle in in synchrony, and the patterns of DNA synthesis and division were similar to those observed in cultures prepared by mitotic selection. Immediately after dilution, the rates of synthesis of RNA and protein increased threefold. This system provides a simple technique for production of large quantities of highly synchronized cells and may ultimately provide information on the biochemical mechanisms regulating cell-cycle traverse.     

Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. VII. Hormonal control of DNA synthesis and its possible significance to the problem of liver regeneration

The initiation of DNA synthesis has been studied in quiescent primary cultures of fetal rat hepatocytes using defined hormones and chemically defined medium. Preparations of crystalline insulin (0.01–10 µg/ml) or 20,000-fold purified somatomedin (0.01–1 µg/ml) are stimulatory. Growth hormone (0.025 µg/ml) and hydroxycortisone (0.025 µg/ml), 3'':5''-GMP! (10^-5 M) fail by themselves to initiate DNA synthesis but added together with insulin, enhance the stimulatory response by 50–100%. Thyroid hormones (L-T₃, L-T₄, 7.5–30 ng/ml) are by themselves without effect, but when they are added to thyroid hormone-depleted serum, the reconstituted mixtures show an enhanced capacity to initiate DNA synthesis. In contrast, glucagon (0.01 µg/ml) inhibits the insulin-stimulated response by about 50% without reducing basal DNA synthesis rates. The results described here and in the accompanying two reports indicate that environmental control over the initiation of DNA synthesis is complex, and can involve the participation of many factors. The in vitro findings are consistent with the concept that liver regeneration is hormonally controlled and raise the possibility that alterations of the intrahepatic ratio of insulin to glucagon are growth regulatory.     

High-resolution radioautography of phlorizin-3H in rings of hamster intestine

Quantitative light microscope radioautographs of galactose-³H and phlorizin-³H were prepared from freeze-dried plastic-embedded hamster small intestine incubated in vitro. The usual uphill epithelial cell accumulation of galactose accompanied by a somewhat smaller lamina propria accumulation was observed in control tissue incubated 3 min in 1 mM galactose-³H. The addition of 5 x 10^-4 M phlorizin to the medium blocked uphill accumulation, but did not prevent galactose equilibration with the epithelial cells. The galactose content of the lamina propria was considerably less than the galactose content of the epithelial cell. Varying the phlorizin-³H content of the medium from 0.6 to 60 µM revealed a brush border binding of phlorizin which followed a Langmuir adsorption isotherm with a half-saturation constant of 13 µM and a maximum binding of 84 µmoles of phlorizin/liter of microvilli or 2.6 x 10⁶ sites/epithelial cell. The phlorizin content of the epithelial cell compartment, excluding microvilli, never exceeded 10% that of the medium after 20 min of incubation. These findings directly support the view that phlorizin is a nontransported inhibitor which binds glucose-galactose carriers at the surface of epithelial cell microvilli.     

Equilibrium and Kinetic Properties of the Interaction between Tetrodotoxin and the Excitable Membrane of the Squid Giant Axon

Squid giant axons were treated with tetrodotoxin (TTX) in concentrations ranging from 1 nM to 25 nM and the resulting decrease in sodium current was followed in time using the voltage clamp technique. The removal of TTX from the bathing solution produced only partial recovery of the sodium current. This suggests that the over-all interaction is more complex than just a reversible reaction. By correcting for the partial irreversibility of the decrease in sodium current, a dissociation constant of 3.31 x 10^-9 M was calculated for the reaction between TTX and the reactive site of the membrane. The data obtained fit a dose-response curve modified to incorporate the correction for partial irreversibility when calculated for a one-to-one stoichiometry. The fit disagreed with that calculated for a reaction between two molecules of TTX with a single membrane-reactive site, but neither supported nor disproved the possibility of a complex formed by two reactive sites with one molecule of TTX. Values of the rate constants for the formation and dissociation of the TTX-membrane complex, k ₁ and k ₂, respectively, were obtained from the kinetic data. The values are: k ₁ = 0.202 x 10⁸ M ^-1, and k ₂ = 0.116 min^-1. The magnitude of the dissociation constant derived from these values is 5.74 x 10^-9 M, which has the same order of magnitude as that obtained from equilibrium measurements. Arrhenius plots of the rate constants gave values for the thermodynamic quantities of activation.     

Thymidine transport by cultured Novikoff hepatoma cells and uptake by simple diffusion and relationship to incorporation into deoxyribonucleic acid

The initial rate of thymidine-³H incorporation into the acid-soluble pool by cultured Novikoff rat hepatoma cells was investigated as a function of the thymidine concentration in the medium. Below, but not above 2 µM, thymidine incorporation followed normal Michaelis-Menten kinetics at 22°, 27°, 32°, and 37°C with an apparent K_m of 0.5 µM, and the V_max values increased with an average Q₁₀ of 1.8 with an increase in temperature. The intracellular acid-soluble ³H was associated solely with thymine nucleotides (mainly deoxythymidine triphosphate [dTTP]). Between 2 and 200 µM, on the other hand, the initial rate of thymidine incorporation increased linearly with an increase in thymidine concentration in the medium and was about the same at all four temperatures. Pretreatment of the cells with 40 or 100 µM p-chloromercuribenzoate for 15 min or heat-shock (49.5°C, 5 min) markedly reduced the saturable component of uptake without affecting the unsaturable component or the phosphorylation of thymidine. The effect of p-chloromercuribenzoate was readily reversed by incubating the cells in the presence of dithiothreitol. Persantin and uridine competitively inhibited thymidine incorporation into the acid-soluble pool without inhibiting thymidine phosphorylation. At concentrations below 2 µM, thymidine incorporation into DNA also followed normal Michaelis-Menten kinetics and was inhibited in an apparently competitive manner by Persantin and uridine. The apparent K_m and K_i values were about the same as those for thymidine incorporation into the nucleotide pool. The over-all results indicate that uptake is the rate-limiting step in the incorporation of thymidine into the nucleotide pool as well as into DNA. The cells possess an excess of thymidine kinase, and thymidine is phosphorylated as rapidly as it enters the cells and is thereby trapped. At low concentrations, thymidine is taken up mainly by a transport reaction, whereas at concentrations above 2 µM simple diffusion becomes the principal mode of uptake. Evidence is presented that indicates that uridine and thymidine are transported by different systems. Upon inhibition of DNA synthesis, net thymidine incorporation into the acid-soluble pool ceased rapidly. Results from pulse-chase experiments indicate that a rapid turnover of dTTP to thymidine may be involved in limiting the level of thymine nucleotides in the cell.     

The Site of Calcium Binding in Relation to the Activation of Myofibrillar Contraction

Skeletal muscle myofibrils, in the presence of 2 mM MgCl₂ at pH 7.0, were found to have two classes of calcium-binding sites with apparent affinity constants of 2.1 x 10⁶ M ^-1 (class 1) and ∼3 x 10⁴ M ^-1 (class 2), respectively. At free calcium concentrations essential for the activation of myofibrillar contraction (∼10^-6 M) there would be significant calcium binding only to the class 1 sites. These sites could bind about 1.3 µmoles of calcium per g protein. Extraction of myosin from the myofibrils did not alter their calcium-binding parameters. Myosin A, under identical experimental conditions, had little affinity for calcium. The class 1 sites are, therefore, presumed to be located in the I filaments. The class 1 sites could only be detected in F actin and myosin B preparations which were contaminated with the tropomyosin-troponin complex. Tropomyosin bound very little calcium. Troponin, which in conjunction with tropomyosin confers calcium sensitivity on actomyosin systems, could bind 22 µmoles of calcium per g protein with an apparent affinity constant of 2.4 x 10⁶ M ^-1. In view of the identical affinity constants of the myofibrils and troponin and the much greater number of calcium-binding sites on troponin it is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.     

The metabolism of histone fractions. VI. Differences in the phosphorylation of histone fractions during the cell cycle

Phosphorylation of histone fractions in the presence and absence of DNA synthesis was measured using the new “isoleucine-limiting” method for synchronizing Chinese hamster cells in early G₁-phase. Using preparative electrophoresis, histone f1 phosphorylation was found to be dependent upon cell-cycle position, being absent in G₁-arrested and G₁-traversing cells and active in the S-phase. The absence of f1 phosphorylation in G₁-arrested cells, which are known to exhibit f1 turnover, indicates that f1 phosphorylation is not an obligatory part of the f1 turnover process. In contrast to histone f1, it was found that histone f2a2 phosphorylation is independent of cell-cycle position, occurring with equal magnitude in the G₁-traversing state when DNA synthesis is essentially absent and in the S-phase when DNA synthesis is active. When cells were arrested in the G₁-state by isoleucine deprivation, f2a2 phosphorylation continued to be active, occurring at 56% of the rate observed in the G₁-traversing state. These results indicate that phosphorylation of histone f2a2 is independent of f2a2 synthesis, independent of DNA synthesis, and independent of histone f1 phosphorylation. Because f2a2 is actively phosphorylated in G₁-arrested cells known to be active in the synthesis of various types of RNA (including messenger) as well as in G₁-traversing and S-phase cells, we feel that phosphorylation of histone f2a2 should continue to be considered in models concerning activation of DNA template activity.     

Cholinesterase activity per unit surface area of conducting membranes

According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm² surface of sensory axons of the walking leg of lobster is 1.2 x 10^-3 µM/hr. (σ = ± 0.3 x 10^-3; SE = 0.17 x 10^-3); the corresponding value for the motor axons isslightly higher: 1.93 x 10^-3 µM/hr. (σ = ± 0.41 x 10^-3; SE = ± 0.14 x 10^-3). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 µM/hr. (σ = ± 73.5; SE = ± 32.6) versus 111.6 µM/hr. (σ = ± 28.3; SE = ± 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10^-4 µM/mm²/hr. (σ = ± 0.96 x 10^-4; SE = ± 0.4 x 10^-4). (3) The Ch-esterase activity per mm² surface of squid giant axon is 9.5 x 10^-5 µM/hr. (σ = ± 1.55 x 10^-5; SE = ± 0.38 x 10^-5). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10^-5 µM/mm²/hr. (σ = ± 3.24 x 10^-5; SE = ± 0.93 x 10^-5). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm² per impulse.     

Nature of the Schwann cell electrical potential. Effects of the external ionic concentrations and a cardiac glycoside

The effects on the Schwann cell electrical potential of external ionic concentrations and of K-strophanthoside were investigated. Increasing (K)_o depolarized the cell. The potential is related to the logarithm of (K)_o in a quasi-linear fashion. The linear portion of the curve has a slope of 45 mv/ten-fold change in (K)_o. Diminutions of (Na)_o and (Cl)_o produced only small variations in the potential. Calcium and magnesium can be replaced by 44 mM calcium without altering the potential. Increase of (Ca)_o to 88 mM produced about 10 mv hyperpolarization. The cell was hyperpolarized by 11 mv and 4 mv within 1 min after applying K-strophanthoside at concentrations of 10^-3 and 10^-5 M, respectively. No variations of cellular potassium, sodium, or chloride were observed 3 min after applying the glycoside. The hyperpolarization caused by 10^-3 M K-strophanthoside was not observed when (K)_o was diminished to 1 or 0.1 mM or was increased to 30 mM. At a (K)_o of 30 mM, 10^-2 M strophanthoside was required to produce the hyperpolarizing effect. In high calcium, the cell was further hyperpolarized by the glycoside. The initial hyperpolarization caused by the glycoside was followed by a gradual depolarization and a decrease of the cellular potassium concentration. The results indicate that the Schwann cell potential of about -40 mv is due to ionic diffusion, mainly of potassium, and to a cardiac glycoside-sensitive ion transport process.     

Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.     

Action of colcemid in sea urchin eggs

The effects of Colcemid, the deacetyl-N-methyl derivative of colchicine, on the eggs of Arbacia punctulata were investigated. Colcemid in concentrations of 2.7 x 10^-5 M or greater blocks syngamy (the fusion of the pronuclei) in these eggs. Although a tenfold decrease in concentration of Colcemid usually permits the pronuclei to fuse, the subsequent division is blocked. In the sea urchin egg, the duration of presyngamy is about 15 min during which time there is no DNA synthesis. However, DNA synthesis is recorded in Colcemid-blocked cells prior to syngamy. Radioautographs of Colcemid-blocked cells which were immersed into thymidine-³H exhibited silver grains above each of the pronuclei. The action of Colcemid on Arbacia eggs is reversible. Nevertheless, exposures to 2.7 x 10^-5 M Colcemid for only 3 min, initiated 5 min after insemination, caused delays of 70 min in subsequent division. In general, cells are more sensitive to Colcemid prior to the time when the mitotic spindle is being assembled than at presyngamy stages. The results are discussed in terms of Colcemid action on pronuclear fusion and cell division.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

MAINTENANCE OF IMAGINAL DISCS OF DROSOPHILA MELANOGASTER IN CHEMICALLY DEFINED MEDIA

A phosphate-buffered saline and a chemically defined synthetic medium for in vitro maintenance of imaginal discs of Drosophila melanogaster were developed. The composition of the chemically defined medium was varied in order to optimize the incorporation of tritiated uridine into RNA and tritiated amino acids into acid-insoluble protein. The optimal ranges obtained were: pH, 6.75–7.35; osmolarity, 285–345 milliosmoles/liter; sodium concentration, 40–60 mM/liter; potassium concentration, 40–60 mM/liter; magnesium concentration, 0.5–3.5 mM/liter; calcium concentration, 0.3–1.5 mM/liter; and inorganic phosphate concentration, 1.5–4.0 mM/liter. The phosphate-buffered saline is superior to a commonly used insect Ringer solution in maintaining total RNA and acid-insoluble protein synthesis in culture. The chemically defined synthetic medium permits linear total RNA and acid-insoluble protein synthesis for more than 48 hr, DNA synthesis for several hours, normal differentiation to occur after 74 hr in vitro, and trypsinization of imaginal discs into single cell suspensions without developmental damage.     

Effects of the Intracellular Ca Ion Concentration upon the Excitability of the Muscle Fiber Membrane of a Barnacle

The membrane excitability and contraction were examined in single barnacle muscle fibers with different internal Ca⁺⁺ concentrations by using buffer solutions made up with EGTA and Ca-gluconate in various proportions. During the passage of dc currents the membrane shows all-or-none spike potentials for internal Ca⁺⁺ concentrations below about 8 x 10^-8 M, oscillatory potential changes in the range between 8 x 10^-8 to 5 x 10^-7 M, but neither oscillatory nor spike potentials were seen for concentrations above 5 x 10^-7 M. All-or-none spike potentials were suppressed when the internal Mg⁺⁺ concentration exceeded 5 mM. The suppression threshold of the internal Ca⁺⁺ concentration for the Sr spike is much higher than that for the Ca spike. The threshold concentration of internal Ca⁺⁺ for contraction was about 8 x 10^-7 M.     

Amino Acid and Sugar Transport in Rabbit Ileum

L-Alanine and 3-O-methyl-D-glucose accumulation by mucosal strips from rabbit ileum has been investigated with particular emphasis on the interaction between Na and these transport processes. L-Alanine is rapidly accumulated by mucosal tissue and intracellular concentrations of approximately 50 mM are reached within 30 min when extracellular L-alanine concentration is 5 mM. Evidence is presented that intracellular alanine exists in an unbound, osmotically active form and that accumulation is an active transport process. In the absence of extracellular Na, the final ratio of intracellular to extracellular L-alanine does not differ significantly from unity and the rate of net uptake is markedly inhibited. Amino acid accumulation is also inhibited by 5 x 10^-5 M ouabain. 3-O-methyl-D-glucose accumulation by this preparation is similarly affected by ouabain and by incubation in a Na-free medium. The effects of amino acid accumulation, of ouabain, and of incubation in a Na-free medium on cell water content and intracellular Na and K concentrations have also been investigated. These results are discussed with reference to the two hypotheses which have been suggested to explain the interaction between Na and intestinal nonelectrolyte transport.