Inhibition of pinocytosis in rat yolk sac by trypan blue.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.     

Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor.

The effects of serum, dissolved oxygen (DO) concentration, and medium pH on hybridoma cell physiology were examined in a controlled batch bioreactor using a murine hybridoma cell line (167.4G5.3). The effect of serum was also studied for a second murine hybridoma cell line (S3H5/gamma 2bA). Cell growth, viability, cell density, carbohydrate and amino acid metabolism, respiration and energy production rates, and antibody production rates were studied. Cell growth was enhanced and cell death was decreased by increasing the serum level. The growth rates followed a Monod-type model with serum being the limiting component. Specific glucose, glutamine, and oxygen uptake rates and specific lactate and ammonia production rates did not change with serum concentrations. Amino acid metabolism was slightly influenced by the serum level. Cell growth rates were not influenced by DO between 20% and 80% air saturation, while the specific death rates were lowest at 20-50% air saturation. Glucose and glutamine uptake rates increased at DO above 10% and below 5% air saturation. Cell growth rate was optimal at pH 7.2. Glucose and glutamine uptake rates, as well as lactate and ammonia production rates, increased above pH 7.2. Metabolic rates for glutamine and ammonia were also higher below pH 7.2. The consumption or production rates of amino acids followed the glutamine consumption very closely. Cell-specific oxygen uptake rate was insensitive to the levels of serum, DO, and pH. Theoretical calculations based on experimentally determined uptake rates indicated that the ATP production rates did not change significantly with serum and DO while it increased continually with increasing pH. The oxidative phosphorylation accounted for about 60% of total energy production. This contribution, however, increased at low pH values to 76%. The specific antibody production rate was not growth associated and was independent of serum and DO concentrations and medium pH above 7.20. A 2-fold increase in specific antibody production rates was observed at pH values below 7.2. Higher concentrations of antibody were obtained at high serum levels, between 20% and 40% DO, and at pH 7.20 due to higher viable cell numbers obtained.     

Kinetic studies on hybridoma cells immobilized in fixed bed reactors

Cultures with immobilized hybridoma cells were performed in fixed bed systems. "Steady state" values for volume-specific substrate uptake and metabolite production rates were determined at various perfusion rates and superficial flow velocities of the medium within the carrier matrix. Data from fixed bed volumes between 50 and 600 ml did not show any difference. The volume-specific glutamine and glucose uptake rate turned out to be independent of the superficial flow velocity, but decreased with decreasing glutamine and glucose concentration. The volume-specific oxygen uptake rate increased with increasing superficial flow velocity and substrate concentration, respectively. A similar behavior was observed for the ratio between oxygen and glucose uptake rate. The production rate for monoclonal antibodies was neither affected by the substrate concentration nor by the superficial flow velocity. The metabolic parameters of the immobilized cells were put into kinetic equations and compared to those of suspended cells. It could be concluded that the metabolism of the immobilized cells is determined by the oxygen supply within the macroporous carriers. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 535-541, 1997.     

Mass spectrometry: A tool for on-line monitoring of animal cell cultures

The magnetic sector mass spectrometer is able to detect oxygen uptake and carbon dioxide production rates from animal cell cultivations performed in 101 biorectors. Such data have not been available with the use of classic exhaust gas analysis applying paramagnetic analyzers and infra-red sensors due to the insensitivity of the apparatus available. In the course of the present work we were able to demonstrate, that the oxygen uptake rate correlates to the number of viable cells. Additionally oxygen uptake rates supplied on-line information about the actual physiology of the cells: When the rates changed during the cultivation process, this immediately indicated the occurrence of limitations of components in the medium. The information could be useful in timing key events, such as performing splits or harvesting the bioreactor.Abbreviations OUR oxygen uptake rate - CDPR carbon dioxide production rate - RQ respiratory quotient This publication is dedicated to the 65 ^th birthday of Prof. Dr. F. Wagner, University of Braunschweig.     

Control of L-phenylalanine production by dual feeding of glucose and L-tyrosine

Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated. A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured. Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 0.1 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensor.In a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway. Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreased.The average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding. Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period. During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value. The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy. (c) 1996 John Wiley & Sons, Inc.     

Heterotrophic activity,glucose uptake and primary productivity in Lake Kinneret*

SUMMARY. Chlorophyll, ATP and glucose concentrations as well as glucose uptake rate and primary production were determined every 2–4 h during three 24–h surveys in Lake Kinneret. In spite of the fluctuations in glucose uptake rate, the estimation of the daily glucose uptake rate from a single sampling carried out at 10.00 hours is justified. The same applies also for the primary production. Routine bi-weekly determinations of primary production and glucose uptake rate were carried out from October 1976 to July 1977. No correlations were found between glucose uptake rates and ambient concentrations of ATP, chlorophyll and glucose. The glucose uptake rate showed two distinct seasonal patterns; the period which was dominated by the Peridinium (January-April) and the rest of the year. In the former period the percentage of glucose-carbon utilized out of the carbon formed in the water column by the photosynthetic activity, fluctuated between 1 and 9%, and in the latter period fluctuated between 5 and 37%. The yearly average was 11.1%, SD 9.8.     

Chemically Characterized Media for Study of Foot-and-Mouth Disease Virus in Baby Hamster Kidney Cells

Foot-and-mouth disease virus can be grown in baby hamster kidney cells with a chemically characterized medium containing only tris(hydroxymethyl)-amino-methane (Tris) buffer, glucose, glutamine, and salts. Virus infectivity was only 0.5 log unit less than in a complex cell growth medium containing serum, tryptose phosphate, and lactalbumin hydrolysate. At high multiplicity of infection, production was maximal in 5 hr, with the virus remaining largely intracellular. Glucose and glutamine appeared to act independently of each other although both were required at about the same time during the virus production cycle. Glutamine had the greater effect and could not be replaced by amino acids, purines, and pyrimidines. Glutamine also stimulated cellular oxygen uptake in both normal and infected cells. Serum and other organic components added singly to the defined medium did not increase the virus yield. Studies on uninfected cells over a 5-hr incubation period showed that the defined medium maintained protein and ribonucleic acid synthesis at rates similar to the complex cell growth medium. These rates were much lower in media containing only inorganic salts and Tris buffer. Glucose, however, was more important to uninfected cellular metabolism than was glutamine. Defined medium containing dialyzed calf serum produced the highest rate of protein synthesis.     

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture

Summary Recombinant human interferon- production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation. Offprint requests to: P. M. Hayter     

NITROGEN UTILIZATION AND METABOLISM RELATIVE TO PATTERNS OF N2 FIXATION IN CULTURES OF TRICHODESMIUM NIBB1067

We measured uptake kinetics for four combined N sources, ambient rates of N uptake and N₂ fixation, glutamine synthetase activity (transferase and biosynthetic), and concentrations of intracellular pools of glutamate (glu) and glutamine (gln) in cultures of Trichodesmium NIBB1067. N dynamics and metabolism were examined to assess the relative importance of N₂ fixation and N uptake to Trichodesmium nutrition. Comparisons were made between cultures grown on medium without added N, with excess NO, or with excess urea. Of the combined N sources tested, Trichodesmium NIBB1067 had the highest affinity for NH; high uptake capacities for NH, urea, and glu; and little capacity for NO uptake. In cultures grown on medium without added N, NH accumulated in the medium during growth, resulting in high NH uptake rates relative to rates of N₂ fixation. Glu uptake rates were low but consistent throughout the diel period. In cultures grown on excess NO or urea, uptake of these compounds supplied the majority of the daily N demand, although some N₂ fixation occurred during the light period. NO uptake rates were reduced when N₂-fixation rates were high. In all of the cultures, the highest gln/glu ratios and the lowest glutamine synthetase transferase/biosynthetic ratios were observed during the period when rates of total N uptake were highest. In cultures growing exponentially on medium without added N, N₂ fixation accounted for 14%– 16% of the total daily N uptake. Uptake of NH and glu, presumably regenerated within the culture vessels, represented 84%–86% of the daily N uptake. Because these systems were closed, net growth was constrained by the rate at which N₂ could be fixed into the system. However, total daily N turnover was greater than that necessary to accommodate the observed increase in culture biomass. The rapid N turnover rates observed in these cultures may support gross productivity and balance the high rates of C fixation observed in natural populations of Trichodesmium.     

Nutrition of eidamella deflexa. IV. Rate of utilization of sugar and amino nitrogen compared with rate of growth and pigment elaboration

Summary The rate of utilization byEidamella deflexa of non-protein nitrogen and dextrose equivalent (reducing sugar) was investigated in each of two media, and compared with rates of growth and pigment elaboration. In a glycine-maltose basal medium the depletion curves for nitrogen and D.E. followed a similar pattern, with the lowest point on the curves corresponding to the period of maximum production of cellular material. With autolysis of cells there was a definite increase in D.E. and a slight increase in amino nitrogen in the medium. In a peptone-sucrose medium both D.E. and amino nitrogen were removed from the medium at a more or less similar and constant rate, but the final amount of each remaining in the medium after eleven days was considerably less than that in the glycinemaltose medium although the latter medium permitted greater growth and pigment elaboration. There seemed to be no correlation between rates of utilization of nitrogen and sugar and the period of maximum growth in the sucrose-peptone medium. In neither medium was there an apparent relation between the rate of onset of pigment production and the rate of utilization of nitrogen and sugar.     

Methods for increasing monoclonal antibody production in suspension and entrapped cell cultures: biochemical and flow cytometric analysis as a function of medium serum content.

The growth and antibody production of the SP2/0-derived hybridoma HB124 (ATCC) grown in media containing varying amounts of fetal bovine serum (FBS) were monitored using biochemical and flow cytometric methods. Hybridomas grown in 100 ml spinner flasks with RPMI-1640 containing varying amounts of serum demonstrated that cell growth, viability and IgG production show significant changes when serum content is decreased from 10.0 to 5.5 to 1.0 and 0.5%. A longer lag phase resulted when the lower serum content media were used. Cellular rates of glucose uptake showed a significant increase as serum levels were lowered. Similarly, exponential phase IgG production rates increased as the amount of serum was decreased, probably as a result of the decreased rate of exponential growth. Flow cytometric analysis showed a similar increase in cellular IgG content as medium serum levels declined. In contrast, the maximum IgG concentrations were found in flasks containing 1% FBS or above with the lowest concentration in the 0.5% FBS flask being due to the lower numbers of viable cells. Cells grown in microporous hollow fiber reactors were fed with medium containing serum which was decreased stepwise with time. Decreasing medium serum content stepwise from 10 to 2.5% resulted in increased antibody production. However, complete removal of serum from the medium resulted in a significant drop in antibody productivity. Cumulative antibody production was equivalent for cells grown entirely in medium containing 10% FBS and for those which experienced a drop to 2.5% FBS. To compare a defined serum-free medium preparation with medium containing 10% FBS, cells were again grown in batch suspension culture and analyzed. The growth rates were similar but there was a significant difference in IgG production rates. The serum-free culture exhibited both higher cellular production rates and higher IgG concentrations. These results indicate that decreasing medium serum content can adversely affect antibody yield because of lower cell viabilities, not because of lower production rates. Use of a defined serum-free medium, as done in this study, results in higher yields because of a higher IgG production rate as well as good cell growth and viability.     

Sugar metabolic analysis of suspensions of plant cells using an FT-IR/ATR method

A simple, rapid and accurate evaluation of the sugar uptake rate of suspended plant cells from culture media was developed with the predicted sugar contents measured by mid-infrared spectroscopy using a Fourier transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance (ATR) accessory. We performed plant cell cultivation with Nicotiana tabacum cv. Bright Yellow No.2 (TBY-2) in culture media, which had various combinations of glucose, fructose and sucrose concentrations at the initial stage, and measured simultaneously each sugar content in the medium by the FT-IR/ATR method. By applying a logistic function to the predicted sugar contents and cell density in the medium during cultivation, the specific sugar uptake rates by the suspended TBY-2 cells were easily and continuously obtained. Thus the kinetic sugar uptake phenomena by the TBY-2 cells were well confirmed overall using the developed method. Additionally it was found that the fraction of sucrose of the initial total sugar content might kinetically affect the sugar uptake process and cell growth. Also, the relationship between the nondimensional cell density and sucrose content could be classified into three groups on the basis of the initial fraction of sucrose.     

Determination of structural regions important for Ca(2+) uptake activity in Arabidopsis MCA1 and MCA2 expressed in yeast

MCA1 is a plasma membrane protein that correlates Ca(2+) influx and mechanosensing in Arabidopsis. MCA2 is a paralog of MCA1, and both share 72.7% amino acid sequence identity and several common structural features, including putative transmembrane (TM) segments, an EF hand-like region in the N-terminal half, a coiled-coil motif in the middle and a PLAC8 motif in the C-terminal half. To determine structural regions important for Ca(2+) uptake activity, the activity of truncated forms of MCA1 and MCA2 was assessed using yeast expression assays. The N-terminal half of MCA1 with a coiled-coil motif (MCA1(1-237)) did not have Ca(2+) uptake activity, while MCA2(1-237) did. The N-terminal half of MCA1 without the coiled-coil motif (MCA1 (1-185)) showed Ca(2+) uptake activity, as did MCA2(1-186). Both MCA1(1-173) and MCA2(1-173) having the EF hand-like region had Ca(2+) uptake activity. Deletion of a putative TM segment (Ile11-Ala33) and the Asp21 to asparagine mutation in MCA1 and MCA2 abolished Ca(2+) uptake activity. Finally, MCA1(173-421) and MCA2(173-416) lacking the N-terminal half had no Ca(2+) uptake activity. These results suggest that the N-terminal half of both proteins with the EF hand-like region is necessary and sufficient for Ca(2+) uptake and that the coiled-coil motif regulates MCA1 negatively and MCA2 positively.     

Growth and nutrient dynamics of beech (Fagus sylvatica L.) seedlings in acid soils

The relative uptake rates of N, P, K, S, Ca, Mg, Fe, Mn, Zn, Cu, and Al were estimated in beech seedlings pot cultured in the field in six acid soils (treatments). The relative uptake rates were compared with the relative growth rates. The relative uptake rates of N, K and Ca agreed well with the growth rates of the seedlings irrespective of widely differing soil conditions (acid sand-clayey till, pH 4–6). The relative uptake rates of P, Fe, and Al differed from that predicted by the growth rate. The uptake rates of Fe and Al were highest at the lowest growth rates, and the P uptake rate was lower than the growth rate in these treatments. Thus the P availability probably limited growth in an eluvial (E) horizon of a podzol, and possibly in the illuvial (B) horizon of a podzol and in an acid clayey till (Dystric Cambisol). Low P uptake was associated with a tendency towards higher relative root growth rates. In terms of the concept of steady state nutrition the high relative root growth rate in some treatments may be interpreted as an acclimation to low P supply. The P limitation seemed to be related to interactions among Fe, Al and organic compounds of the soil solution.FAX no: +4646104423     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.     

Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

The production of the extracellular alkaline protease Savinase (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum at a dilution rate between 0.14 and 0.17 h(-1), whereas the yield of Savinase on glucose was found to increase with decreasing specific growth rate. A linear relationship between the ribosomal RNA content and the specific production rate was found, indicating that the translational capacity may be limiting for product formation. The dynamics of the production of Savinase were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific growth rate. Furthermore, the adaptation to high glucose concentrations was faster at high dilution rates than at low dilution rates.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (aw) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the aw of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 aw. The maintenance coefficients for glucose uptake were similar at both aw values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 aw.     

Kinetic Analysis of Aspergillus oryzae Cultivations on Starch

Cultivation of Aspergillus oryzae on starch is described as a combination of two rate processes: Starch hydrolysis and the cellular activities of the fungi including growth, enzyme production and maintenance. Kinetic models are presented to describe growth, enzyme production, starch hydrolysis and uptake of the hydrolysis products. Numerical values of the model parameters indicated that the rate controlling step of A. oryzae growth on starch was not starch hydrolysis, but the substrate uptake process. Glucose was one of the starch hydrolysis products. About 35% of the substrate consumed for biomass synthesis was glucose. Its accumulation in the medium did not cause repression of the starch hydrolysing enzymes. Steady state starch hydrolysis rates increased with initial starch concentration in the medium. Starch hydrolysing enzymes of A. oryzae have extensive industrial uses. This study may help in a more detailed understanding of the kinetic aspects of the production of these enzymes.     

Urea production and accumulation in the green toad, Bufo viridis

The toad, Bufo viridis , can live for several months without access to free water, absorbing soil-bound water down a water-potential gradient created, mainly, by accumulating urea in its body fluids. We investigated if the retention of urine was sufficient to account for the rate of accumulation or if an increased rate of urea production was needed in order to do so. The basal rate of urea production in unfed animals in the absence of osmotic stress was estimated by two methods; first, analysis of the bathing medium and, secondly, collection and analysis of urine at two-hourly intervals. This was then repeated with animals fed a weight-maintaining diet. Generally similar results were obtained by either method in both fed and unfed animals, although higher urea production rates were found in the former. Although it had been planned to apply the short interval method to toads with free access to water, the control condition for toads transferred to soil, it proved to be impracticable. Some animals did not bathe for almost a day, during which time minute quantities of urine were obtained. Larger volumes were only produced during or after bathing. Consequently, animals which were partially immersed in water were substituted as controls. Total urea content was determined in these and in toads after a week on soil. The calculated increase was compared to that which could be expected from urine retention. It was found that urea accumulated at more than twice the predicted rate. When rates of accumulation were calculated over longer periods, urine retention alone was sufficient to account for them within three weeks on soil, the usual period required for acclimation. We concluded that B. viridis increased its rate of urea production only for a short period, until a favourable water potential gradient was achieved.     

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.