Inhibition of thymidine uptake in asynchronous HeLa cells by nitrobenzylthioinosine

Nitrobenzylthioinosine (NBMPR), an inhibitor of nucleoside transport by human erythrocytes, was found to be a potent inhibitor of thymidine uptake by asynchronous monolayer cultures of HeLa cells. Rates of thymidine uptake by the cultures at 20 °C were constant between 10 and 40 sec after thymidine addition and were assayed during this interval; TTP was the principal metabolite of thymidine and the thymidine phosphates accumulated at constant rates which extrapolated through time zero. The lack of an effect of NBMPR on thymidine kinase activity, or on the relative proportions of thymidine metabolites in cell extracts, indicated that NBMPR inhibited thymidine transport. When mediated entry (transport) was eliminated by 2 μM NBMPR, a significant diffusional component of thymidine entry was apparent. The mediated component of thymidine uptake exhibited Michaelis-Menten kinetics and apparent K_m and V_max values of 0.5 μM and 10–21 pmoles/min/10⁶ cells were obtained. When NBMPR-treated cells were transferred to NBMPR-free medium, inhibition of thymidine uptake persisted, suggesting that NBMPR was firmly bound to the transport inhibitory sites.     

Evidence that calmodulin may be involved in phytohaemagglutinin-stimulated lymphocyte division

Phytohaemagglutinin-stimulated and non-stimulated incorporation of [³H]thymidine into human peripheral blood lymphocytes is inhibited by the calcium antagonist PY 108–068 and by the calmodulin antagonists trifluo-perazine andN-(6-aminohexyl)-5-chloro-l-naphthalene sulphonamide (W7). It is argued that calmodulin may be involved in both non-stimulated [³H]thymidine uptake in lymphocytes and also in the lymphocyte response to phytohaemagglutinin.     

Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [³H]thymidine incorporation, ¹⁴CO₂ uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 10⁹ to 2.4 × 10⁹ cells liter⁻¹ and 7 to 47 μg of C liter⁻¹, respectively. The average bacterial cell volume was 0.185 μm³. [³H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10⁻¹² to 200 × 10⁻¹² mol liter⁻¹ h⁻¹. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter⁻¹ h⁻¹. Bacterial production estimates from [³H]thymidine incorporation and ¹⁴CO₂ uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [³H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the ³H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the ¹⁴CO₂ uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.     

Bacterial productivity in the water column and sediments of the Georgia (USA) coastal zone: Estimates via direct counting and parallel measurement of thymidine incorporation

Three methods of estimating bacterial productivity were compared using parallel samples of Atlantic Ocean water (within 0.25–15 km of the Georgia coast). The frequency-of-dividing cells (FDC) method and the [³H]thymidine incorporation method gave results which were strongly correlated (r=0.97), but the FDC estimates were always higher (X2 to X7) than the [³H]thymidine estimates. Estimates of bacterial productivity ranged from 2–4×10⁸ cells·l^–1·h^–1 at 0.25 km from shore to 1–9×10⁷cells·l^–1·h^–1 at 15 km. A method involving incubation of 3-m filtrates and direct counting gave results that could not be easily translated into estimates of bacterial productivity. Application of the FDC method to sediment samples gave high productivity estimates, which could be not reconciled with productivity estimates based on sediment oxygen uptake.     

Methyl mercury uptake by free and immobilized cyanobacterium

Methyl mercury uptake in free cells and different immobilizates of the cyanobacteriumNostoc calcicola has been examined. The general growth of the immobilized cyanobacterial cells could be negatively correlated with methyl mercury uptake. Alginate spheres proved most efficient in terms of uptake rate (0.48 nmol mg protein^–1 min^–1, 10 min) and total bioaccumulation (10.71 nmol mg protein^–1, 1 h) with a bioconcentration factor of 3.3×10³. Alginate biofilms showed a faster methyl mercury accumulation rate (0.83 nmol mg protein^–1 min^–1, 10 min) with a saturation of 10.28 nmol mg protein^–1 reached within only 30 min (bioconcentration factor, 3.1×10³). Foam preparations with a slow initial uptake approximated biofilms but were characterized by a lower bioconcentration factor (2.8×10³). Free cells, in comparison, maintained the initial slow rate of uptake (0.62 nmol mg protein^–1 min^–1, 10 min), saturating at 30 min (8.81 nmol mg protein^–1), and the resultant lowest bioconcentration factor (2.7×10³). Cell ageing (30 days) brought a drastic reduction (3-fold) in organomercury uptake by free cells while alginate spheres maintained the same potential. Foam preparations of the same age showed a significant improvement in methyl mercury uptake followed by only a marginal decline in alginate biofilms. Data are discussed in the light of the physiological efficiency and longevity of immobilized cells.     

Thymidine uptake by developing sea urchin embryos

Unfertilized Paracentrotus lividus eggs accumulate very little thymidine. Upon fertilization, however, uptake increases sharply. The pool for thymidine and/or its metabolic products is saturated after 40 min of exposure. Its size is expandable and proportional to the initial concentration of thymidine in the medium. The uptake rate is low shortly after fertilization, increases until 40 min after fertilization and remains constant thereafter. Of the radioactivity taken up in the form of [³H]thymidine during a 30 min exposure beginning at 60 min after fertilization, about 1% is associated with the acid-insoluble fraction and 99% with the acid-soluble fraction.     

Preparation of vinyl thymidine ester catalyzed by protease and its chemical polymerization

Transesterification reaction of 0.25 M thymidine with 1 M divinyladipate in dimethylformamide (DMF) was catalyzed by an alkaline protease (5 mg ml^–1) from Streptomyces sp. (20 units mg^–1 min) at 30 °C for 7 days to give 5-O-vinyladipoyl thymidine (yield 77%) without formation of any by-products. Poly(vinyl alcohol) containing thymidine branches could be obtained by its free-radical polymerization.     

Zinc uptake by isolated rat hepatocytes

Isolated rat hepatocytes were used to investigate the uptake of zinc at early exposure times. Hepatocytes were incubated with ⁶⁵Zn (1–500 μM) and samples were withdrawn at times ranging from 25 s to 60 min. A biphasic pattern of uptake was observed with a rapid first phase of uptake followed by a slower second phase. The relationship between velocity of uptake and substrate concentration for the first phase was nonlinear, while that of the second phase was linear. The presence of 10 μM cadmium produced a decrease in the velocity of uptake of only the first phase. This suggests that the first phase is at least partly carrier mediated, while there is no indication of involvement of a carrier in the second phase. KCN (1 mM) and carbonyl cyanide m-chlorophenylhydrazone (2 μM), did not cause any change in the uptake of ⁶⁵Zn (1 μM), which suggests that there is no active component in the uptake of zinc.     

Autoradiographic and ultrastructural study of Cucurbita pepo root cells during their growth and differentiation

Summary Cortex cells of the root meristem of Cucurbita pepo (0.0–0.5 mm from the cap junction), in the 3–4, 5–6 and 7–8 mm segments above the root tip, and the cells of the first three layers of lateral part of root cap were the object of the present study. The volume of cortex cells increases more than 20 times in the 7–8 mm segment as compared with meristematic cells, and the volume of cytoplasm about sevenfold. The largest increment of the cytoplasmic volume occurs between 0.5–6.0 mm. In consecutive root segments the sustained increase of the volume of nuclei takes place. By applying autoradiography the following processess have been investigated: DNA synthesis (³H thymidine uptake), template activity of DNA (³H actinomycin D(³H AMD)-binding), RNA synthesis (³H uridine incorporation), and protein synthesis (³H leucine). In the root cap cells and in segments where meristematic activity is over, DNA is replicated by endomitosis. On the basis of nuclear labelling it appears that nuclei in the 3–4 mm segment reach 4C ploidy state, but in the 7–8 mm segment half of the nuclei reach the 8C ploidy state. Most of the root cap cells are 4C, the remaining cells are 8C. Considering the uptake of ³H thymidine into nucleoli one may suppose that in the root cap cells nucleolar DNA is underreplicated, and to a lesser degree in 5–6 and 7–8 mm segments, while in 3–4 mm segment DNA is overreplicated as compared to meristem cells. Measurements of nucleolar volume, ³H uridine uptake, ³H AMD binding and quantity of granular component, indicate that the most noticeable nucleolar activity takes place in meristematic zone and in root parts showing the highest increase of cytoplasmic volume (3–4 and 5–6 mm segments). ³H leucine is still incorporated intensely into 7–8 mm segment, in which the concentration of ribosomes is low, however they are present in the form of polysomes. Comparison of ³H thymidine uptake into nuclear DNA with ³H AMD binding and ³H uridine incorporation into nuclei indicates that endomitotic DNA replication results in an increase of DNA template activity in root cap cells as well as in 3–4 and 5–6 mm segments; in the 7–8 mm segment binding of ³H AMD slightly decreases, while ³H uridine incorporation is considerably reduced. Divergence between the ploidy state, ³H AMD binding and ³H uridine incorporation can be due to the increment of the condensed chromatin area in differentiated cells. Plastids and mitochondria reach full maturity in 3–4 mm segment. The increasing volume density of ER and diminishing volume density of Golgi structures is accompanied by differentiation of cortex cells.This work was partly supported by Polish Academy of Sciences, Botanical Committee, Grant 217/II     

Zooplankton induced decrease in inorganic phosphorus uptake by plankton in an oligotrophic lake

Experiments conducted on samples collected from a large oligotrophic lake revealed the following: (1) excretion rates of PO _inf4 ^sup3– by single Daphnia thorata were below detection (5 pmol animal^–1 min^–1) in 20 ml of oligotrophic lake water over a period of 10 min, (2) experimental addition of D. thorata to 20 ml aliquots of lake water decreased community-wide microbial uptake of PO _inf4 ^sup3– on two occasions (as measured by ³²PO _inf4 ^sup3– incorporation), and (3) the presence of D. thorata increased uptake by organisms smaller than 1µm, and decreased uptake by large phytoplankton. The specific mechanism for these responses remains unclear, but the results imply that when phytoplankton larger than 1µm encounter cm scale patches of water recently occupied by Daphnia they may experience decreased PO _inf4 ^sup3– availability rather than elevated concentrations of PO _inf4 ^sup3– caused by excretion. We show that ³²P uptake experiments using natural plankton assemblages can be influenced by the presence or absence of large zooplankton, and that neither grazing, turbulence, nor PO _inf4 ^sup3– excretion can account for this influence.     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Calculation of Cell Production from [3H]Thymidine Incorporation with Freshwater Bacteria

The conversion factor for the calculation of bacterial production from rates of [³H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20°C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 × 10¹⁸ cells mol^-1 of thymidine incorporated into the trichloroacetic acid (TCA) precipitate (standard error = 0.29 × 10¹⁸; n = 54), when the generation time exceeded 20 h. At generation times of <20 h, the average conversion factor was 11.8 × 10¹⁸ cells mol^-1 of thymidine incorporated into TCA precipitate (standard error = 1.72 × 10¹⁸; n = 47). The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20°C than at 15 and 10°C. A detailed examination of the [³H]thymidine concentrations that were needed to achieve maximum labeling in DNA was carried out 6 times during a complete growth cycle. During periods with low generation times and high conversion factors, 15 nM [³H]thymidine was enough for the maximum labeling of the TCA precipitate. This suggests that incorporation of [³H]thymidine into DNA is probably limited by uptake during periods with generation times of <20 h and that freshwater bacterioplankton cell production sometimes is underestimated when a conversion factor of 2.15 × 10¹⁸ cells mol^-1 of thymidine incorporated is used.     

Clearance rates of bacteria by the rotifer Filinia longiseta (Ehrb.) measured using three tracers

The clearance rates (CRs) of bacteria by Filinia longiseta were measured at 19°C, both in situ in Lake Loosdrecht and in the laboratory during summer. The tracer particles used in the field were: (1) 0.51 µm fluorescent microspheres, and (2) fluorescently labelled bacteria (FLB). A third type of tracer particle, natural [methyl-³H]-thymidine-labelled bacteria (< 1.2 µm), were used as a radiotracer in a laboratory experiment. The uptake of the first two tracer-particle types was measured by microscopic examination of the rotifer guts. In the third case, the uptake of radioactivity was determined by liquid scintillation counting. The rate of uptake of the microspheres decreased 10 min after the start of the experiment, probably because the gut passage time was exceeded. Using a 5 min feeding time, the rate of uptake of microspheres was higher than that of the FLB, though the variation in the uptake in both cases was high. The ingestion rates and CRs of bacteria by F. longiseta based on the fluorescent tracers were: microspheres, 5115 bact.ind^–1 h^–1 and 0.368 µl ind^–1 h^–1; FLB, 2252 bact.ind^–1 h^–1 and 0.162 µl ind^–1 h^–1. The mean CR using the thymidine-labelled natural bacteria and a 10 min feeding time was 0.179 µl ind^–1 h^–1. Thus, the CR based on the microsphere method was twice as high as for the other two methods.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

Characteristics of plankton communities in Chinese integrated fish ponds: effects of excessive grazing by planktivorous carps on plankton communities

Biomass and production of plankton communities were investigated in two Chinese integrated fish culture ponds in August, Dianshanhu Pond (with high density of planktivorous carp) and Pingwang Pond (with low density of planktivorous carp). The plankton communities were composed of rotifers, protozoans, phytoplankton (<40 µm) and bacteria. The large phytoplankton (>40 µm), cladocerans and copepods were rare because of grazing pressure by the carp. The density or biomass of bacteria (1.93 × 10⁷ and 2.20 × 10⁷ cells ml^–1 on average in Dianshanhu and Pingwang Ponds, respectively), picophytoplankton (24.6 and 18.5 mg m^–3 Chla on average) and rotifers (5372 and 20733 ind. 1^–1 on average) exceeded the maximum values reported for natural waters.The average [³H]thymidine uptake rates were 694 and 904 pmoles 1^–1 h^–1 (13.4 and 20.6 µgC 1^–1) and the bacterial production by the >2 µm fraction amounted 21–28% of total [³H] thymidine uptake rate in both ponds. The mean chlorophylla concentrations were 59.1 and 183 mg m^–3 in Dianshanhu and Pingwang Ponds, respectively. 82.4% and 65.3% of the total Chla was contributed by the <10 µm nano- and picophytoplankton in each pond, respectively. In particular, the picophytoplankton contribution amounted 41.2% of thtal Chla in Dianshanhu Pond. Primary production was 2.5 and 3.4 gC m^–2 d^–1 in each pond, respectively, and >50% of production was contributed by picophytoplankton. The mean biomasses of protozoa were 168 µg 1^–1 and 445 µg 1^–1 and those of rotifers were 763 µg 1^–1 and 1186 µg 1^–1 in Dianshanhu and Pingwang Ponds, respectively. The ecological efficiencies expressed in terms of the ratios of primary production to zooplankton production were 0.22 and 0.31, for the two ponds.     

l-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled l-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [³H]l-serine and 10 nM of [³H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 °C, for different periods up to 30 min. The uptake of [³H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na⁺, K⁺ or choline ions. At all postnatal ages studied, [³H]-GABA uptake showed a high activity in the presence of Na⁺ ions and at 30 °C. The values of K_m were 90–489 μM in l-serine uptake. However, in the uptake of GABA the values of K_m were 80–150 μM. The highest values of V_max were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

In situ measurement of fine root water absorption in three temperate tree species—Temporal variability and control by soil and atmospheric factors

Miniature heat balance-sap flow gauges were used to measure water flows in small-diameter roots (3–4 mm) in the undisturbed soil of a mature beech–oak–spruce mixed stand. By relating sap flow to the surface area of all branch fine roots distal to the gauge, we were able to calculate real time water uptake rates per root surface area (J_s) for individual fine root systems of 0.5–1.0 m in length. Study aims were (i) to quantify root water uptake of mature trees under field conditions with respect to average rates, and diurnal and seasonal changes of J_s, and (ii) to investigate the relationship between uptake and soil moisture θ, atmospheric saturation deficit D, and radiation I. On most days, water uptake followed the diurnal course of D with a mid-day peak and low night flow. Neighbouring roots of the same species differed up to 10-fold in their daily totals of J_s (<100–2000 g m⁻² d⁻¹) indicating a large spatial heterogeneity in uptake. Beech, oak and spruce roots revealed different seasonal patterns of water uptake although they were extracting water from the same soil volume. Multiple regression analyses on the influence of D, I and θ on root water uptake showed that D was the single most influential environmental factor in beech and oak (variable selection in 77% and 79% of the investigated roots), whereas D was less important in spruce roots (50% variable selection). A comparison of root water uptake with synchronous leaf transpiration (porometer data) indicated that average water fluxes per surface area in the beech and oak trees were about 2.5 and 5.5 times smaller on the uptake side (roots) than on the loss side (leaves) given that all branch roots <2 mm were equally participating in uptake. Beech fine roots showed maximal uptake rates on mid-summer days in the range of 48–205 g m⁻² h⁻¹ (i.e. 0.7–3.2 mmol m⁻² s⁻¹), oak of 12–160 g m⁻² h⁻¹ (0.2–2.5 mmol m⁻² s⁻¹). Maximal transpiration rates ranged from 3 to 5 and from 5 to 6 mmol m⁻² s⁻¹ for sun canopy leaves of beech and oak, respectively. We conclude that instantaneous rates of root water uptake in beech, oak and spruce trees are above all controlled by atmospheric factors. The effects of different root conductivities, soil moisture, and soil hydraulic properties become increasingly important if time spans longer than a week are considered.     

Specific binding of progesterone to the cell surface and its role in the meiotic divisions in Rana oocytes

Progesterone is believed to act at the cell surface to induce the resumption of the meiotic divisions in amphibian oocytes. Analysis of [³H]- and [¹⁴C]progesterone uptake and exchange by the plasma-vitelline membrane complex, nucleus and cytoplasm of the isolated Rana oocyte indicates that progesterone uptake by the plasma membrane is saturable, specific and temperature-dependent, and has a slow off-rate. Estradiol (a noninducer) did not compete with progesterone, whereas testosterone (an inducer) blocked progesterone uptake by the membrane complex. Scatchard-type plots indicate an apparent K_d of 5.1·10⁻⁷ M over the [progesterone]_o range of 0.01–1.0 μM with maximum binding at about 70 fmol per oocyte. Membrane uptake at higher [progesterone]_o (2–40 μM) indicates apparent cooperative binding, with saturation up to 10 pmol per oocyte. Cytoplasmic uptake was apparently nonspecific and less temperature-dependent than membrane uptake and steroid concentrations (progesterone and pregnanediones) exceeded water solubility by 30–60 min. Nuclear uptake was saturable and specific but uptake was independent of temperature. A comparison of membrane binding and a physiological response (nuclear breakdown) indicated only about 10% of the membrane sites need be filled to initiate a 50% response.     

A glycine betaine transport system in Aphanothece halophytica and other glycine betaine-synthesising cyanobacteria

Uptake of exogenous ¹⁴C-glycine betaine has been followed in the cyanobacterium Aphanothece halophytica and other species able to synthesise glycine betaine in response to osmotic stress. At 1 mmol dm^–3 uptake was rapid (flux rate=29.50 nmol m^–2 s^–1), equilibrating at an internal concentration of 120 mmol dm^–3 within 30 min. This rapid uptake, coupled with high internal accumulation, was characteristic of glycine betaine-synthesising cyanobacteria only. The ¹⁴C-glycine betaine transported was not catabolised. Kinetic studies indicated a Michaelis-Menten type relationship (K _m=2.0 mol dm^–3, V _max=45 nmol min^–1 mm^–3 cell volume), with a pH optimum of 8.0–8.5. Darkness dramatically decreased the flux rate. Higher ¹⁴C-glycine betaine levels occurred in cells growth in medium of elevated osmotic strength, and glycine betaine uptake was sensitive to changes in external salinity. A relationship between Na⁺ availability and glycine betaine uptake was observed, with >80 mmol dm^–3 Na⁺ required for optimal stimulation of uptake in seawater-grown cells. Severe hyperosmotic stress (1000 mmol dm^–3 NaCl) reduced the rate of glycine betaine uptake but increased internal glycine betaine concentration at equilibrium. Hypo-osmotic stress caused a decline in the internal glycine betaine concentration due to an increased rate of loss, indicating that the efflux system was also sensitive to ambient salinity changes. It is envisaged that this active transport system may be an adaptive mechanism in halophilic glycine betaine-synthesising cyanobacteria.