Induced alteration in uptake properties of Tetrahymena and its association with the development of mating competency.

The rate of uridine uptake in Tetrahymena declines by an order of magnitude by two hours after shiftdown to a non-nutrient buffer. This alteration in uptake properties cannot be accounted for by an increase in the intracellular pool of uridine, an increase in apparent Km for uptake or a decline in the rate in which uridine is processed intracellularly. It is argued that the decrease in uridine uptake is due to a reduction in numbers of functional transport molecules exposed at the cell surface and is a reflection of a developmentally related cell surface transformation. In addition, the putative decline in functional transport molecules cannot be entirely explained by metabolic turnover of these molecules in the absence of replacement, nor does it require the synthesis of new protein. We discuss the possibility that a shift in equilibrium between accessible and inaccessible transporters is operating. Finally, a close correlation between conditions which elicit the transport alteration and those which allow the development of mating competency suggests that the two phenomena may be coordinately regulated.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. IV. Nucleoside transport in cells depleted of nucleotides by treatment with KCN

Incubation of Novikoff rat hepatoma cells in glucose-free basal medium containing 2 mM KCN results in a rapid and almost complete loss of uracil and adenine nucleotides. By following the fate of radioactivity from ³H-nucleoside pulse-labeled cells during incubation with KCN it was shown that the nucleotides are degraded to nucleosides and bases which are released into the culture fluid. Depletion of the cells of nucleotides by incubation with KCN allows a direct analysis of the kinetics of uridine transport into the cell, since KCN-treated cells fail to phosphorylate uridine. Uridine uptake follows normal Michaelis-Menten kinetics with an apparent K_n of about 50 μm at 18°C. Uptake is by facilitated diffusion since it does not require energy and uridine is not transported against a concentration gradient. The effects of KCN are largely prevented by the presence of 10 mM glucose in the medium. They are also rapidly reversed by resuspending the cells in fresh medium without KCN. Upon removal of KCN, the cells rapidly regenerate their nucleotide pools and resume growth at the normal rate.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

Uridine uptake and its intracellular phosphorylation during the cell cycle

The rate of 5-³H uridine uptake into Chinese hamster V79 cells and the rate of its incorporation into RNA increase tenfold during the cell cycle. Both reactions exhibit the same apparent K_m(1.7 × 10^?5 M ). Chromatography of acid-soluble material from cells incubated with 5-³H uridine (0.25 μM) at different times of the cell cycle revealed that intracellular uridine was rapidly phosphorylated at all times, even though cells in late S and G₂ take up roughly ten times as much uridine as cells in G₁. Uridine kinase activity in synchronized cells increases about two and one-half-fold during the same time period, and in exponentially growing cells is not saturated until the external uridine concentration is raised above 200 μM. It is concluded that the change in uridine kinase activity during the cell cycle is not responsible for the tenfold increase in the rate of uridine transport, and that these two processes are independently regulated.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent K_m value of 46 μM and a V_max of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na⁺ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na⁺ was related to the transmembraneous Na⁺-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Microcalorimetric Study on the Toxic Effect of Pb<Superscript>2+</Superscript> to <Emphasis Type="Italic">Tetrahymena</Emphasis>

The toxic effect of Pb²⁺ has been studied in eukaryotic cells by using Tetrahymena as a target. The maximum power (P _m) and the growth rate constant (k) were determined, which showed that values of P _m and k were linked to the concentration (C) of Pb²⁺. The addition of Pb²⁺ caused a decrease of the maximum heat production and growth rate constant, indicating that Tetrahymena growth was inhibited in the presence of Pb²⁺, and Pb²⁺ took part in the metabolism of cells. From micrographs, morphological changes of Tetrahymena were observed with addition of Pb²⁺, indicating that the toxic effect of Pb²⁺ derived from destroying the membrane of surface of Tetrahymena. According to the thermogenic curves and photos of Tetrahymena under different conditions, it is clear that metabolic mechanism of Halobacterium halobium R1 growth has been changed with the addition of Pb²⁺.     

Uridine transport and RNA synthesis in growing and in density-inhibited animal cells

Both chick embryo fibroblasts and mouse 3T3 cells reduce the rate at which they incorporate H³ uridine into RNA as their growth becomes inhibited at high cell density. This reduction occurs as a function of the cell population density, and with chick embryo cells (in contrast to 3T3 cells) it is not accompanied by significant medium alterations. This indicates the importance of the cell population density in the control of cellular metabolism. The decline in H³ uridine incorporation is paralleled by a decline in the rate of uptake of the isotope into the acid-soluble pool, suggesting that decreased entry of H³ uridine into the cell, rather than a decreased rate of RNA synthesis, is responsible for the reduced rate of incorporation into RNA of density-inhibited cells. This suggestion was confirmed by finding that when the restriction on uridine uptake was overcome by increasing the concentration of uridine in the medium, the density-dependent inhibition of uridine incorporation was largely reversed. We conclude that, even though the rate of H³ uridine incorporation into RNA is reduced three- to five-fold in density-inhibited cells, the rate of synthesis of pulse-labeled RNA continues at 70 to 85% of the rapidly-growing rate.     

DNA Synthesis Cycle in Blepharisma*

SYNOPSIS. Observations made with tritiated thymidine uptake indicate that Blepharisma cannot utilize this precursor directly from the organic medium. An intermediary, such as a food organism (bacteria or Tetrahymena, is needed. These food organisms are not only capable of incorporating thymidine from the medium, but must also break it down or prepare it in a form that is utilizable for Blepharisma. DNA synthesis phase in Blepharisma occurs 17 hours after division. Maximum incorporation is obtained 18 and 19 hours postdivision and lasts thru 24 hours. This is followed by a 1–2 hour postsynthesis phase and division.     

RNA synthesis during spermiogenesis in the sea urchin: a high resolution autoradiographic study of [3H]uridine incorporation

Summary

RNA synthesis has been studied during spermiogenesis of Paracentrotus lividus by high resolution autoradiography using [³H]uridine as a labeled precursor. Under the experimental conditions used [³H]uridine incorporation is detectable only at the early spermatid stage. Labeling is distributed mainly over the nucleus and it appears completely absent from the mitochondria. After RNase digestion radioactivity falls to a background level, demonstrating that RNA synthesis actually occurs in early spermatids of P. lividus. It follows that in P. lividus during spermiogenesis cell differentiation is not entirely dependent on stored premeiotic RNAs.     

Reversible arrest of Chinese hamster V79 cells in G2 by dibutytyl AMP.

Mouse L cells 929 were cloned in supplemented Eagle's minimal medium enriched with lactalbumin and yeast extract and buffered with HEPES. Multiplication was followed photographically in single clones from the 8-cell stage through 6–7 days. Addition of the folic acid analogue methotrexate (amethopterin) in 5 × 10^?6 M concentration slowed growth only after two cell generations; 10^?4 M uridine had no effect on growth except when combined with methotrexate. The two agents together blocked cell division quickly and symptoms of thymine-less death developed in few days. The cells could be rescued before 48 h by removal of the inhibitors, or by addition of folic acid or thymidine. The combination of methotrexate with uridine blocks DNA synthesis in Tetrahymena by inhibition of thymidylate synthesis and of thymidine uptake from the complex medium. Apparently the same mechanisms operate in L cells grown in a complex medium containing thymidine.     

UPTAKE OF GUANINE BY THE DIATOM,PHAEODACTYLUM TRICORNUTUM1

Nitrate-cultured cells of Phaeodactylum tricornutum Bohlin lack the ability to take up guanine but can do so after a period of nitrogen deprivation, i.e. photosynthesis in nitrogen-free medium. Maximum rate of uptake occurred after 24 h of nitrogen deprivation. The development of ability to take up guanine required CO₂ fixation and was prevented by cycloheximide, ammonium or nitrate. The guanine taken up accummulated in the cells almost entirely as a compound which is probably methylated hypoxanthine. Guanine uptake was dependent upon metabolism and exhibited Michaelis-Menten like kinetics with a half-saturation value of 0.48 ± 0.05 μM guanine and a maximum uptake rate for guanine of ca. 200 nmol · 10^?8 cells · h^?1. Rate of uptake increased hyperbolically with Na⁺ concentration, with 8.25 mM Na⁺ supporting half-maximal rate, and it was inhibited by K⁺ ions.     

Low concentrations of dimethyl sulphoxide accelerate conjugation and incorporation of glycine and glucosamine intetrahymena

Summary Dimethyl sulphoxide at relatively low comentrations, 0.01 to 1 mM, enhanced the conjugation and cell-to-cell adhesion of complementary strains of matingTetrahymena thermophila. The time required to form stable conjugates was reduced by dimethyl sulphoxide. This chemical stimulated the uptake of glycine and glucosamine from the suspending media. Incorporation of 2-¹⁴C-glycine and 6-³H-D-glucosamine into protein and glycoprotein was enhanced in whole cells, surface membrane and cilia. Incorporation of glucosamine into the microsomal fraction was increased in the dimethyl sulphoxidetreated cells while there was little change in glycine incorporation. There were no detectable changes in glycine and glucosamine incorporation into the nuclear fractions isolated from conjugatingTetrahymena exposed to dimethyl sulphoxide.     

Uptake of l-Phenylalanine in Synchronous Chlorella fusca. Characterization of the Uptake System

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a K_m= 5 μM. V_max, was 0.1 nmol/min × 10⁷ cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l -phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l -phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L-phenylalanine. Variations in the ratio of Na4⁺ to K⁺ in the external solution during uptake experiments did not have any influence upon the uptake rate of l -phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4-Dinitro-phenol inhibited the uptake completely. Exchange between internal and external l -phenylalanine could not be demonstrated. The K_m value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l -phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells.     

Transport du sulfate à travers la double membrane limitante, ou enveloppe, des chloroplastes d'épinard

Evidence is presented for low rates of carriermediated uptake of sulphate, thiosulphate and sulphite into the stroma of the C3 plant Spinacia oleracea. Uptake of sulphate in the dark was followed using two techniques (1) uptake of sulphate [³⁵S] as determined by silicon oil centrifugal filtration and (2) uptake as indicated by inhibition of CO₂-dependant O₂ evolution rates after addition of sulphate.Sulphate, thiosulphate and sulphite were transported across the envelope leading to an accumulation in the chloroplasts. Sulphate transport had saturation kinetics of the Michaelis-Menten type (Vmax : 25 μmoles . mg⁻¹ chl . h⁻¹ at 22°C ; Km : 2.5 mM). The rate of transport for sulphate was not influenced either by illumination or pH change in the external medium. Phosphate was a competitive inhibitor of sulphate uptake by chloroplasts (Ki : 0.7 mM, fig. 1). The rate of transport for phosphate appeared to be much higher than for sulphate. When the chloroplasts were pre-loaded with labelled sulphate, radioactivity was rapidly released after addition of phosphate into the external medium. Consequently, the transport of sulphate occurs by a strict counter-exchange : for each molecule of sulphate entering the chloroplast, one molecule of phosphate leaves the stroma, and vice-versa.The uptake of sulphate by isolated intact chloroplasts exchanging for internal free phosphate induced a lower rate of photophosphorylation, which in turn inhibited CO₂-dependent O₂ evolution.The presence, on the inner membrane of the chloroplast envelope, of a specific sulphate carrier, distinct from the phosphate translocator, is discussed.     

Uridine transport in Novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis.

Time courses of [³H]uridine uptake as a function of uridine concentration were determined at 25° in untreated and ATP-depleted wild-type and uridine kinase-deficient Novikoff cells and in mouse L and P388 cells, Chinese hamster ovary cells and human HeLa cells. Short term uptake was measured by a rapid sampling technique which allows sampling of cell suspensions in intervals as short as one and one-half seconds. The initial segments of the time courses were the same in untreated, wild-type cells in which uridine is rapidly phosphorylated and in cells in which uridine phosphorylation was prevented due to lack of ATP or uridine kinase. The initial rates of uptake, therefore, reflected the rate of uridine transport. Uridine uptake, however, was approximately linear for only five to ten seconds at uridine concentrations from 20–160 μM and somewhat longer at higher concentrations. In phosphorylating cells the rate of uridine uptake (at 80 μM) then decreased to about 20–30% of the initial rate and this rate was largely determined by the rate of phosphorylation rather than transport. At uridine concentrations below 1 μM, however, the rate of intracellular phosphorylation in Novikoff cells approached the transport rate. The apparent substrate saturation of phosphorylation suggests the presence of a low K_m uridine phosphorylation system in these cells. The “zero-trans” (zt) K_m for the facilitated transport of uridine as estimated from initial uptake rates fell between 50 and 240 μM for all cell lines examined. The zero-trans V_max values were also similar for all the lines (4–15 pmoles/μ1 cell H₂O.sec). The time courses of uridine uptake by CHO cells and the kinetic constants for transport were about the same whether the cells were propagated (and analyzed for uridine uptake) in suspension or monolayer culture. When Novikoff cells were preloaded with 10 μM uridine the apparent K_m and V_max values (infinite-trans) were two to three times higher than the corresponding zero-trans values. Uridine transport was inhibited in a simple competitive manner by several other ribo- and deoxyribonucleosides. All nucleosides seem to be transported by the same system, but with different efficiencies. Uridine transport was also inhibited by hypoxanthine, adenine, thymine, Persantin, papaverin, and o-nitrobenzylthioinosine, and by pretreatment of the cells with p-chloromercuri-benzoate, but not by high concentrations of cytosine, D-ribose or acronycin. The inhibition of uridine transport by Persantin involved changes in both V and K. Because of the rapidity of transport, some loss of intracellular uridine occurred when cells were rinsed in buffer solution to remove extracellular substrate, even at 0°. This loss was prevented by the presence of a transport inhibitor, Persantin, in the rinse fluid or by separating suspended cells from the medium by centrifugation through oil. Metabolic conversion of intracellular uridine were also found to continue during the rinse period. The extent of artifacts due to efflux and metabolism during rinsing increased with duration of the rinse.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

An autoradiographic method of detectingA. laidlawii andM. hyorhinis in cell cultures

Summary The autoradiographic investigation of L cells and Chinese hamster cells for the presence of mycoplasmas (A. laidlawii andM. hyorhinis) using uridine/uracil (UdR/U) testing is a rapid and reliable method suitable for the serial checking of even a small number of cells. It depends on a reduced incorporation of [³H]uridine and an increased uptake of [³H]uracil into the RNA of mycoplasma-infected cells, shown in autoradiograms by the density of the grains and their distribution. Results obtained by the autoradiographic technique correspond approximately to specific activity values of RNA-infected cells after the incorporation of [³H]uridine and [³H]uracil.     

Changes of uridine permeability during the maturation of tunicate eggs.

The time course of uridine uptake by eggs and embryos of the tunicate Ascidia callosa was studied using 5-min pulses of [³H]uridine at intervals from the unfertilized egg to the 16-cell embryo. The unfertilized egg is permeable to uridine, but 5 min after fertilization uptake begins to drop, reaching a minimum of 30% of the unfertilized rate about 30 min after fertilization. At 45 min after fertilization, permeability begins to increase, reaching a plateau about 3 hr after fertilization at the two-cell stage. The initial decrease in permeability occurs at first polar body production; the increase at 45 min is coincident with the formation of the second polar body. Substrate concentration experiments up to 200 μM show strict concentration dependence for uridine uptake. The inhibitors p-chloromercuribenzoate (PCMB), dinitrophenol (DNP), and thymidine have little, if any effect on permeability. Cold (?1°C) and Na⁺-free sea water inhibit uptake 60% during all three developmental stages. The changes in permeability may be indicative of temporary reorganization of the plasma membrane during the fertilization-initiated completion of meiosis.     

Putrescine uptake in saintpaulia petals

Putrescine uptake and the kinetics of this uptake were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments of [³H] or [¹⁴C] putrescine were done on single petals at room temperature at various pH values. The results show that putrescine uptake occurs against a concentration gradient at low external putrescine concentration (0.5-100 micromolar) and follows a concentration gradient at higher external putrescine concentrations (100 micromolar to 100 millimolar). 2,4-Dinitrophenol and carbonylcyanide-m-chlorophenylhydrazone, two uncouplers, had no effect on putrescine uptake. Uptake rates were constant for 2 hours, reaching a maximum after 3 to 4 hours. Putrescine uptake depended markedly on the external pH and two maxima were observed: at low external concentrations of putrescine, the optimum was at pH 5 to 5.5; at higher concentrations the optimum was at pH 8.     

Effect of in vivo prostaglandin treatment on H-PGF2α uptake in hamster corpora lutea

Pregnant hamsters were administered (SC) prostaglandin or vehicle on the morning of the 4th day of pregnancy. Serum progesterone was significantly depressed (p<.01) at 0.5, 2, and 6 hours after treatment with 100 μg PGF_2α. Serum progesterone levels were unchanged 2 hours and 6 hours after treatment with 100 μg PGF_2β and 2 hours after treatment with 1 mg PGF_2β. Progesterone levels were depressed to less than 1 ng/ml 6 hours after treatment with 1 mg PGF_2β. The specific uptake of ³H-PGF_2α in whole hamster corpora lutea was significantly depressed 2 hours and 6 hours following 100 μg PGF_2α treatment. A 15% depression in specific uptake occurred 0.5 hour post-treatment. Treatment with 100 μg PGF_2β resulted in no change. Administration of 1 mg PGF_2β resulted in depressed ³H-PGF_2α uptake at both 2 and 6 hours post-treatment.Prostacyclin (PGI₂) treatment resulted in no change in either ³H-PGF_2α specific uptake or serum progesterone 2 hours after 100 μg treatment SC. These parameters were both reduced approximately 30% 6 hours post-treatment. Treatment with 6-keto-PGF_1α resulted in a complete lack of measurable ³H-PGF_2α uptake and serum progesterone levels less than 1 ng/ml at both 2 and 6 hours after treatment with 1 mg SC.