A new principle of continuous flow cell cultivation

Summary A completely liquid-filled culture chamber with gas exchange across a synthetic membrane (Larsen andNilsson 1985) was incorporated into an automatic continuous flow system. The absence of an airliquid interface in the system permits removal of cell samples, and addition of fresh medium, under strictly sterile conditions. In this system,Tetrahymena pyriformis can be kept under optimal growth conditions in a rich nutrient medium and any defined cell density may be maintained for extended periods of time by varying the dilution rate of the culture. Furthermore, it has been possible to demonstrate, in the slope of the growth curve, even small changes which are difficult to detect in batch cultures since the duration of these changes is short. In the continuous flow system, the relative cell volume distribution and the food vacuole forming capacity of the cells were unaltered; however, all cells contained small refractive granules. The system permits the culture volume to be varied, but a standard volume of 20 ml was maintained in most experiments. Since the culture volume is small, the system requires less than one liter of fresh medium per week to maintain the cells in the exponentially multiplying growth phase.     

Mass spectrometric determination of O2 gas exchange during a dark-to-light transition in higher-plant cells

The exchange of O₂ and CO₂ by photoautotrophic cells of Euphorbia characias L. was measured using a mass-spectrometry technique. During a dark-tolight transition the O₂ uptake rate was little affected whereas CO₂ efflux was decreased by 40%. In order to differentiate eventual superimposed O₂-uptake processes, the kinetics of O₂ exchange resulting from brief illuminations were measured with a highly sensitive device. When the cells were exposed to a saturating light for short periods, the rate of O₂ uptake passed through a series of transients: there was first a stimulation occurring 2–3 s after the appearance of O₂ from water-splitting, followed 30 s later by an inhibition. These two transients were reduced 80% by 3-(3,4-dichlorophenyl)1, 1-dimethylurea (DCMU), indicating that they relied on the linear transport of electrons in the chloroplasts. The first transient (stimulation of an O₂ uptake) was little affected by mitochondrial inhibitors such as antimycin A and oligomycin or the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) but was increased in presence of KCN. When spaced flashes (2 us duration; 100-ms intervals) were used instead of continuous light, this transient was almost suppressed indicating that it was dependent on the saturation of some component of the chloroplastic chain. The second transient (inhibition of O₂ uptake) was present when spaced flashes were used instead of continuous light. It was markedly decreased by addition of CCCP and mitochondrial inhibitors (antimycin A, oligomycin, KCN) which strongly indicates that it relied on mitochondrial respiration. It is concluded from these experiments that illumination of the cells resulted in an inhibition of mitochondrial respiration, but the resulting inhibition of O₂ uptake was hidden by the appearance of an O₂-uptake process of extramitochondrial origin, presumably located in the chloroplast.Abbreviations CCCP carbonylcyanide mchlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Rubisco ri-bulose-1,5-bisphosphate carboxylase/oxygenase The authors thank Drs A. Vermeglio, P. Thibault and P. Gans for helpful discussions.     

Effects of chloramphenicol on the physiology and fine structure ofTetrahymena pyriformis GL: Correlation between diminishing inner mitochondrial membrane and cell doubling

Summary A time-dependent loss of tubular infolding of the inner mitochondrial membrane was reported recently as an effect of the cytostatic drug, methotrexate (MTX), onTetrahymena (Nilsson 1983); this finding was interpreted as an inhibition of mitochondrial protein synthesis. In the present study, the cells were exposed to chloramphenicol (CAP), an inhibitor of mitochondrial translation, at the same concentrations (1–25 mM) as MTX; the question asked was whether the two drugs acted similarly. CAP affected cell proliferation by causing a dose-dependent prolongation of the generation time, but at 10–25 mM permitted only a limited number of cell doublings, whereas 1 mM MTX inhibits growth after 5 cell doublings. With CAP the inner mitochondrial membrane diminished gradually in accordance with the number of cell doublings at 10–25 mM, but in 2 mM CAP, for example, some tubular infoldings were still present after 17 cell doublings. The gradual loss of the inner mitochondrial membrane correlated with a gradual decrease in the cellular ATP content, irrespective of the concentration of the drug but dependent on the progress of the cells through their first cycle when exposed to the drug; in cells which continued to proliferate, the ATP content remained at a value corresponding to 80% of the control value. With respect to cell proliferation, the two drugs act differently. CAP is less toxic than MTX, reflected in a 10 times shorter recovery time for cell proliferation after removal of CAP. Hence, although the structural manifestation of the action of the two drugs on mitochondria is identical, their target site may differ.     

Mass-spectrometric determination of O2 and CO2 gas exchange in illuminated higher-plant cells

In order to estimate photosynthetic and respiratory rates in illuminated photoautotrophic cells of carnation (Dianthus caryophyllus L.), simultaneous measurements of CO₂ and O₂ gas exchange were performed using ¹⁸O₂, ¹³CO₂ and a mass-spectrometry technique. This method allowed the determination, and thus the comparison, of unidirectional fluxes of O₂ and CO₂. In optimum photosynthetic conditions (i.e. in the presence of high light and a saturating level of CO₂), the rate of CO₂ influx represented 75±5% of the rate of gross O₂ evolution. After a dark-to-light transition, the rate of CO₂ efflux was inhibited by 50% whereas the O₂-uptake rate was little affected. The effect of a recycling of respiratory CO₂ through photosynthesis on the exchange of CO₂ gas was investigated using a mathematical model. The confliction of the experimental data with the simulated gas-exchange rates strongly supported the view that CO₂ recycling was a minor event in these cells and could not be responsible for the observed inhibition of CO₂ efflux. On the basis of this assumption it was concluded that illumination of carnation cells resulted in a decrease of substrate decarboxylations, and that CO₂ efflux and O₂ uptake were not as tightly coupled in the light as in the dark. Furthermore, it could be calculated from the rate of gross photosynthesis that the chloroplastic electron-transport chain produced enough ATP in the light to account for the measured CO₂-uptake rate without involving cyclic transfer of electrons around PS I or mitochondrial supplementation.Abbreviations Chl chlorophyll - K_d permeability coefficient The authors thank Drs A. Vermeglio and P. Thibault, Dépt. de Biologie, CEN-Cadarache, St. Paul Lez Durance, France, for helpful discussions.     

Improvement of growth conditions and gas exchange of<Emphasis Type="Italic"> Fagus sylvatica</Emphasis> L. seedlings planted below a recently thinned<Emphasis Type="Italic"> Pinus sylvestris</Emphasis> L. stand

Abstract Gas exchange and growth of beech seedlings planted in the understory of a recently thinned pinewood were recorded for 2 years. Relative irradiance was assessed by hemispherical photographs taken just after the thinning. Predawn water potential (_pd), daily gas exchange and chlorophyll fluorescence were measured several times during the two growing seasons. Maximum values of photosynthesis (A _max) and stomatal conductance to water vapour (g _wvmax) were established from daily data. Maximum quantum efficiency of PS II was recorded at dawn by taking the variable to maximum chlorophyll fluorescence ratio on dark adapted leaves (F _v/F _m). In the middle of each summer, leaf nitrogen content and leaf mass per area were evaluated, and height growth and basal area increment were recorded at the end of the season. The thinning treatment removed half the trees and generated around 10% more available relative irradiance (GLF). This was followed by an increase in net photosynthesis at saturating PPFD (A _sat) and in maximum stomatal conductance to water vapour (g _wvmax). Moreover, specific leaf mass (SLM) and mass based nitrogen content (N_m) showed higher values for seedlings in the thinned stand. In both years, a positive relationship was established between the area based nitrogen content (N_a) and maximum net photosynthesis (A _max). In 1998, a year with a dry summer, seedlings suffered a significant drop in daily A _max irrespective of the thinning regime. This was a response to an increase in stomatal limitation to net photosynthesis, g _wvmax reaching the lowest value on dates with the highest drought. A lack of decrease of Fv/Fm confirmed the absence of significant non-stomatal limitation to A as a consequence of photoinhibition after opening the pinewood. A higher maximum quantum efficiency of open PS II centres (Fv/Fm) was registered in seedlings in the thinned stand. The significance of the differences between the treatments was stronger in the second year after thinning. In 1999, a year with frequent summer storms, water availability increased for seedlings growing under the thinned pinewood. Overall, the reduced pine overstory had a positive effect on physiological responses of beech seedlings, which was translated into improved seedling growth.     

Chamber response time: A neglected issue in gas exchange measurements

When the dimensions of standard commercial chambers for measuring gas exchange cannot accommodate the object being measured, scientists construct their own chambers. The time needed to reach chamber steady state (chamber response time) depends on net system volume (e.g. chamber and tubing volume) and airflow. Unfortunately, some authors take chamber response time into consideration while others ignore it. We present the formula for calculating chamber response time.     

Growth and gas exchange response to water shortage of a maize crop on different soil types

The effect of water shortage on growth and gas exchange of maize grown on sandy soil (SS) and clay soil was studied. The lower soil water content in the SS during vegetative growth stages did not affect plant height, above-ground biomass, and leaf area index (LAI). LAI reduction was observed on the SS during the reproductive stage due to early leaf senescence. Canopy and leaf gas exchanges, measured by eddy correlation technique and by a portable photosynthetic system, respectively, were affected by water stress and a greater reduction in net photosynthetic rate (A _N) and stomatal conductance (g _s) was observed on SS. Chlorophyll and carotenoids content was not affected by water shortage in either condition. Results support two main conclusions: (1) leaf photosynthetic capacity was unaffected by water stress, and (2) maize effectively endured water shortage during the vegetative growth stage.     

Semicontinuous cultivation of photoautotrophic cell suspension cultures in a 20 l airlift-reactor

An airlift-bioreactor system was established for semicontinuous growth of photosynthetically active plant cell suspension cultures in a controlled environment. The bioreactor unit was constructed as a conventional, internal draught tube airlift-reactor, which is characterized by a H D^-1 ratio of 2.9, a ratio of the cross-sectional area of the riser to the cross-sectional area of the downcomer of 0.25 and a surface area of 0.435 m² for illumination. Cultivation experiments could be scaled up to working volumes of maximal 20 1. Sixteen fluorescent tubes were fixed around the outer glass cylinder to provide cells continuously with light. An external cooling device was used to keep the temperature constantly at 27°C. Agitation as well as supply with CO₂ was performed by injecting air enriched with CO₂ through a ring-shaped sparger at the bottom of the vessel. A first set of experiments was carried out with a photoautotrophic culture of Chenopodium rubrum L. Cell material adapted to large scale culture conditions was used to inoculate a modified MS medium (Murashige & Skoog 1962) without any organic constituents. Under these conditions a biomass increase of 1870% was achieved in 18 days. Several physiological parameters (e.g. pigmentation, photosynthetic O₂ evolution, carbohydrate content) were measured routinely to elucidate the growth characteristics of large-scale grown Chenopodium cells. Electron microscopic photographs from different phases of culture growth clearly demonstrate the pattern of cellular development. Special emphasis was placed upon the differentiation of chloroplast ultrastructure. The presented data confirm the feasibility of large-scale culture techniques with photosynthetic active plant cell cultures.Abbreviations D diameter - DW dry weight - FW fresh weight - H height - K_La volumetric oxygen transfer coefficient (h^-1) - MES 2-(N-morpholino)-ethanesulfonic acid - specific growth rate (d^-1) - PAR photosynthetically active radiation (400–700 nm) - Pepcase phosphoenolpyruvate carboxylase - Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase - t_d doubling time (d) - vvm (aeration volume) (medium volume)^-1 min^-1 Dedicated to Prof. F.-C. Czygan on the Occasion of his 60th Birthday     

A new electrical method for the determination of the cell membrane area in plant cells

The membrane are of giant algal cells of Valonia utricularis was determined electrically by using the charge-pulse technique. The membrane was charged to low voltages between 2 and 20 mV by injecting charge pulses of defined amplitude and very short duration (about 100 ns). The injected charge was calculated by measuring the current increment via a potential drop across a 10 resistance in the outer circuit and by considering the preselected charging time. The initial voltage across the membrane was calculated by extrapolation to time zero (=end of the charge pulse). From the values of the injected charge and the voltage built up initially across the membrane, the capacitance of the membrane could be calculated. Assuming that the specific capacity of the two membranes, tonoplast and plasmalemma, arranged in series was 0.5 F cm^-2, the membrane area could be derived from the membrane capacity. The electrically determined membrane area agrees with the geometrically determined one to within 10%.     

Bark stripping in cork oak (Quercus suber L.): effect of an antitranspirant application on gas exchange and water relations of the stripped surface

Quercus suber L. is the primary source of industrial cork, which can be legally collected every 9 years. The main objective of this work was to test the efficiency of an application of an antitranspirant, at three different concentrations, after the bark stripping. For this purpose, several measurements of the gas exchange, water potential, total chlorophylls and the carotenoids contents were determined in cork oak trees, at two times in a day, morning and afternoon. The antitranspirant film was applied immediately after stripping. Transpiration rate showed a significant increase in the afternoon. The parameters, water potential, photosynthetic rates, stomatal conductance and the intrinsic water use efficiency, showed a significant decrease from morning to afternoon. The difference between pigments concentration was not significant throughout the day. Water potential and transpiration rate were high in the treatments with lower antitranspirant concentration. However, the treatment with a higher paraffin concentration showed larger photosynthesis rate. This result suggests that the loss of water observed for the stripping surface can be minimized by a larger concentration of the antitranspirant.     

A new Trichoplusia ni cell line for membrane protein expression using a baculovirus expression vector system

A new cell line, MSU-TnT4 (TnT4), was established from Trichoplusia ni embryos for use with baculovirus expression vectors and evaluated for its potential for membrane protein production. To evaluate membrane protein synthesis, recombinant baculoviruses were constructed to express the human neurotensin receptor 1 as an enhanced green fluorescent protein (GFP) fusion. TnT4 cells had a doubling time of 21 h and expressed the membrane-GFP fusion protein at approximately twice the level as Sf21 cells from the p10 promoter, as evaluated by GFP intensity. Expression of secreted alkaline phosphatase (SEAP) was similar to that of Sf21 cells. Expression of membrane-GFP fusion proteins in recombinant baculoviruses provides a rapid method for evaluating the potential of new cell lines for the production of membrane proteins using a baculovirus expression vector system (BEVS).     

Effects of water stress on gas exchange of field grown <Emphasis Type="Italic">Zea mays</Emphasis> L. in Southern Italy: an analysis at canopy and leaf level

Zea mays is cultivated in the Mediterranean regions where summer drought may lead to photoinhibition when irrigation is not available. In this work the response of maize to water stress was evaluated by gas exchange measurements at the canopy and leaf level. Leaf gas exchange was assessed before, during and after water stress, while canopy turbulent fluxes of mass and energy were performed on a continuous basis. In the early growth period, a linear increment of net ecosystem photosynthetic rate (P _NE) to incoming of photosynthetic photon flux density (PPFD) was found and net leaf photosynthetic rate (P _NL) showed the tendency to saturate under high irradiance. During water stress, the relationship between P _NE and PPFD became curvilinear and both P _NE and P _NL saturated in a range between 1,000 and 1,500 μmol (photons) m⁻² s⁻¹. Leaf water potential (ψ_l) dropped from −1.50 to −1.88 MPa during water stress, indicating that leaf and canopy gas exchanges were limited by stomatal conductance. With the restoration of irrigation, P _NE, P _NL and ψ_l showed a recovery, and P _NE and P _NL reached the highest values of whole study period. Leaf area index (LAI) reached a value of 3.0 m² m⁻². The relationship between P _NE and PPFD remained curvilinear and P _NE values were lower than those of a typical well-irrigated maize crop. The recovery in P _NE and P _NL after stress, and ψ_l values during stress indicate that the photosynthetic apparatus was not damaged while soil moisture stress after-effects resulted in a sub-optimal LAI values, which in turn depressed P _NE.     

Energy requirement for maintenance of the transmembrane potassium gradient in Klebsiella aerogenes NCTC 418: A continuous culture study

Molar growth yield studies on chemostat cultures of Thiobacillus neapolitanus grown in thiosulfate-minerals medium have confirmed earlier observations that the dry weight increased linearly with the dilution rate. The observed increase can be explained neither by a change in cell composition nor by the observed excretion of organic compounds. The increase of the molar growth yield over the full range of growth rates, that is also observed in other obligate chemolithotrophs, was not found in the facultatively chemolithotrophic Thiobacillus A2, grown on thiosulfate or formate. The interpretation of the results in terms of maintenance energy requirement is discussed. It is concluded that these results do not allow a mathematical treatment according to the empirical formula of Pirt.Abbreviation APS Adenosine-5-phosphosulfate     

Higher-plant plasma membrane cytochromeb 561: A protein in search of a function

Summary During the past twenty years evidence has accumulated on the presence of a specific high-potential, ascorbate-reducibleb-type cytochrome in the plasma membrane (PM) of higher plants. This cytochrome is named cytochromeb ₅₆₁ (cytb ₅₆₁) according to the wavelength maximum of its -band in the reduced form. More recent evidence suggests that this protein is homologous to ab-type cytochrome present in chromaffin granules of animal cells. The plant and animal cytochromes share a number of strikingly similar features, including the high redox potential, the ascorbate reducibility, and most importantly the capacity to transport electrons across the membrane they are located in. The PM cytb ₅₆₁ is found in all plant species and in a variety of tissues tested so far. It thus appears to be a ubiquitous electron transport component of the PM. The cytochromesb ₅₆₁ probably constitute a novel class of transmembrane electron transport proteins present in a large variety of eukaryotic cells. Of particular interest is the recent discovery of a number of plant genes that show striking homologies to the genes coding for the mammalian cytochromesb ₅₆₁. A number of highly relevant structural features, including hydrophobic domains, heme ligation sites, and possible ascorbate and monodehydroascorbate binding sites are almost perfectly conserved in all these proteins. At the same time the plant gene products show interesting differences related to their specific location at the PM, such as potentially N-linked glycosylation sites. It is also clear that at least in several plants cytb ₅₆₁ is represented by a multigene family. The current paper presents the first overview focusing exclusively on the plant PM cytb ₅₆₁, compares it to the animal cytb ₅₆₁, and discusses the possible physiological function of these proteins in plants.Abbreviations Asc ascorbate - cyt cytochrome - DHA dehydroascorbate - E₀ standard redox potential - EST expressed sequence tag - His histidine - MDA monodehydroascorbate - Met methionine - PM plasma membrane     

A new set of regulatory molecules in plants: A plant phospholipid similar to platelet-activating factor stimulates protein kinase and proton-translocating ATPase in membrane vesicles

1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, an ether phospholipid from mammals known as platelet-activating factor (PAF), specifically stimulates proton transport in zucchini (Cucurbita pepo L.) microsomes (G.F.E. Scherer, 1985, Biochem. Biophys. Res. Commm. 133, 1160–1167). When plant lipids were analyzed by two-dimensional thin-layer chromatography a lipid was found with chromatographic properties very similar to the PAF (G.F.E. Scherer and B. Stoffel, 1987, Planta, 172, 127–130). This lipid was isolated from zucchini hypocotyls, red beet root, lupin root, maize seedlings and crude soybean phospholipids. It had biological activity similar to that of the PAF, based on phosphorus content, and stimulated the steady-state pH in zucchini hypocotyl microsomes about twofold. Other phospholipids, monoglyceride, diglyceride, triglyceride, oleic acid, phorbol ester, and 1-O-alkylglycerol did not stimulate proton transport. When microsomes were washed the PAF was ineffective but when soluble protein was added the PAF stimulation of H⁺ transport was reconstituted. The soluble protein responsible for the PAF-dependent stimulation of transport activity could be partially purified by diethylaminoethyl Sephacel column chromatography. In the same fractions where the PAF-dependent transport-stimulatory protien was found, a protein kinase was active. This protein kinase was stimulated twofold either by the PAF or by Ca²⁺. When Ca²⁺ was present the PAF did not stimulate protein-kinase activity. When either the PAF, protein kinase, or both were added to membranes isolated on a linear sucrose gradient, ATPase activity was stimulated up to 30%. Comparison with marker enzymes indicated the possibility that tonoplast and plasma-membrane H⁺-ATPase might be stimulated by the PAF and protein kinase. We speculate that a PAF-dependent protein kinase is involved in the regulation of proton transport in plants in vitro and in vivo.Abbreviations BTP 1,3-bis[tris(hydroxymethyl)-methylamino] propane - DEAE diethylaminoethyl - EGTA ethylene glycolbis(-aminoethyl ether)-N,N,N,N,-tetraacetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - PAF platelet-activating - factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine     

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations,rubidium ion,and a cyanine dye as probes

Summary Two of the commonly used probes for measuring membrane potential—lipophilic cations and the cyanine dye diS-C₃(5)—indicated nominally opposite results when tetraphenylarsonium ion was added as a drug to suspensions of metabolizingBacillus subtilis cells. [³H]-Triphenylmethylphosphonium uptake was enhanced by the addition, indicating hyperpolarization, yet fluorescence of diS-C₃(5) was also enhanced, indicating depolarization. Evidence is presented that both effects are artifactual, and can occur without any change in membrane potential, as estimated by⁸⁶Rb⁺ uptake in the presence of valinomycin. The fluorescence studies suggest that tetraphenylarsonium ion displaces the cyanine dye from the cell envelope, or other binding site, into the aqueous phase.The uptake characteristics of the radiolabeled lipophilic cations were quite unusual: At low concentrations (e.g., less than 10 m for triphenylmethylphosphonium) there was potential-dependent uptake of the label to a stable level, but subsequent addition of nonradioactive lipophilic cation caused further uptake of label to a new stable level. Labeled triphenylmethylphosphonium ion taken up to the first stable level could be displaced by 10mm magnesium ion, whereas⁸⁶Rb⁺ uptake was unperturbed. Association of the lipophilic cations with the surface of de-energized cells was concentration-dependent, but there was no evidence for cooperative binding. This phenomenon of stimulated uptake inB. subtilis (which was not seen inEscherichia coli cells or vesicles) is consistent with a two-compartment model with access to the second compartment only being possible above a critical cation concentration. We tentatively propose such a model, in which these compartments are the cell surface and the cytoplasm, respectively.Triphenylmethylphosphonium up to 0.5mm exhibited linear binding to de-energized cells; binding of tetraphenylphosphonium and tetraphenylarsonium was nonlinear but was not saturated at the highest concentration tested (1mm). The usual assumption, that association of the cation with cell surfaces is saturated and so can be estimated on de-energized cells, therefore leads to undercorrected estimates of cytoplasmic uptake inB. subtilis, and hence to overestimates of membrane potential. We describe a more realistic procedure, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells. Using this procedure we estimate the membrane potential inB. subtilis to be 120 mV, inside-negative. The procedure is of general applicability, and should yield more accurate estimates of membrane potential in any system where there is significant potential-dependent binding.Work performed while on sabbatical leave from Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.     

Metabolism of gibberellin A29 in seeds of Pisum sativum cv. Progress No. 9; Use of [2H] and [3H]GAs,and the identification of a new GA catabolite

The metabolism of GA₂₉ in maturing seeds of Pisum sativum cv. Progress No. 9 was further investigated, and the utility of ²H-labelled GAs in conjuction with GC-MS is illustrated. Using [2-²H₁]GA₂₉ as an internal standard, endogenous GA₂₉ was shown to reach a maximal level (ca. 10 g/seed) 27 days from anthesis, and to decline to ca. 1.6 g/seed in mature seeds. In a time-course feed the metabolism of [2-²H₁] [2-³H₁]GA₂₉ applied to 27 day old seeds, and of endogenous GA₂₉, was compared from the ¹H:²H ratios in the recovered GA₂₉. Although both [2-²H₁] [2-³H₁]GA₂₉ and endogenous GA₂₉ were metabolised to the same limited extent to a putative conjugate, in the main metabolic process endogenous GA₂₉ was preferentially converted to an untraceable (i.e. unlabelled) metabolite. In contrast, endogenous GA₂₉ and [1,3-²H₂] [1,3-³H₂]GA₂₉, derived from [1,3-²H₂] [1,3-³H₂]GA₂₀ in a time-course feed, were metabolised in an identical manner. In the latter case isotope loss precluded identification of the metabolite. The structure (8) has been assigned to a GA catabolite present in maturing seeds and seedlings of pea. The isotope data are consistent with this compound being the hitherto untraced metabolite of GA₂₉ in pea.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - M⁺ molecular ion - Me methyl ester - R_T retention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethylsilyl ether     

A new alkalitolerant <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> yeast strain is a promising model for dissecting properties and regulation of Na<Superscript>+</Superscript>-dependent phosphate transport systems

A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na⁺-dependent phosphate transport systems in yeasts. Phosphate uptake by Y. lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na⁺-dependent systems specifically activated by Na⁺. One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate. The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value. The contribution of Na⁺/P_i-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH 9.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1607–1615.Original Russian Text Copyright © 2004 by Zvyagilskaya, Persson.     

A Transplasma Membrane Redox System in Phycomyces blakesleeanus: Properties of a Ferricyanide Reductase Activity Regulated by Iron Level and Vitamin K3

Intact Phycomyces blaskesleeanus mycelia are capable of reducing extracellular ferricyanide and this transmembrane reduction is an enzymatic process, which is enhanced by the presence of 10 mM lactate. It is modulated in response to intracellular iron levels and negatively regulated by iron and copper. It is inhibited by NEM, p CMB, iodoacetate, Zn2+, Cd2+, dicumarol, and capsaicine analog, but not by cloroquine, and activated by Ca2+, Mg2+, Na+, and K+. Ferricyanide reduction was concomitant with proton release into the extracellular medium, both processes being greatly promoted by vitamin K3 following hyperbolic saturation kinetics with regard to ferricyanide concentration. No stoichiometric proton release was observed with regard to ferricyanide reduction in the absence or the presence of vitamin K3. Proton release coupled with ferricyanide reductase activity does not appear to be due to H+-ATPase. The relevance of these findings to the relationship between the two processes is discussed.