Effect of auxins on the formation of ubiquinone by tobacco plant cells in suspension culture

Ubiquinone (UQ) formation in BY-2 tobacco cells was especially promoted by a high concentration of 2,4-D. 2,4,5-T, MCP and NAA also promoted UQ formation in these cells. The UQ content in the cells cultured at high concentrations of 2,4-D was higher than that of controls throughout the culture period. The addition of 2,4-D at an early period in cell growth was very effective in promoting UQ formation, but addition at the stationary phase was ineffective. Cell growth was improved by adding phosphate to the medium but UQ content was decreased. UQ content decreased slowly during subculturing, whereas cell growth recovered gradually.     

Effects of Nutritional Factors on the Formation of Ubiquinone by Tobacco Plant Cells in Suspension Culture

The ubiquinone (UQ) content of BY–2 cells on surface culture was examined to compare with that in suspension culture. The UQ content on surface culture was a little lower than that in suspension culture, but the pattern of the time-course of the UQ formation on surface culture was similar.

The changes of UQ content in BY–2 cells during autolysis were also examined. UQ in the cells subjected to autolysis was not rapidly metabolized nor excreted into the medium.

In order to obtain basic information for UQ formation by BY–2 cells in suspension culture, the cultural conditions, especially nutritional ones were investigated. Addition of 2,4-D was remarkably effective on UQ formation and a higher UQ content was observed with a higher 2,4-D concentration. Sucrose and glucose concentrations in the original medium were also influential factors. The UQ content tends to increase with the decrease of the sugar content. Precursors of UQ, amino acids, vitamins and organic acids were not effective on the UQ formation.     

Characteristics of Cultured Tobacco Cell Strains Producing High Levels of Ubiquinone-10 Selected by a Cell Cloning Technique

Characteristic differences were examined between tobacco cell strains producing high levels of ubiquinone (UQ) and the original tobacco cell line (Nicotiana tabacum L. cv BY-2). The growth rate of strains producing high levels of UQ was about half of that of the original cells. The maximum yield in cell dry weight was about two-thirds of that of original cells. The time-course of UQ formation by selected strains and the respiratory rates were similar to those in the original cells. The UQ contents were much higher than those in original cells, not only per g-dry weight but also per cell. Most UQ in the selected strains were also localized in mitochondria, as well as in the original cells. On protein basis, the yield of purified mitochondria from strains producing high levels of UQ was 4.3 times as much as that from original cells. UQ formation per mg of mitochondrial protein and the molar ratios of UQ to the other electron transport components in selected cells were similar to those in original cells. The ratio of the mitochondrial protein yield in strains producing high levels of UQ to the yield in original cells correlated closely with the ratio of UQ content per g-dry weight in UQ-producing strains to UQ content in original cells.     

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N₂ fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.     

Effects of ethanol on Saccharomyces cerevisiae as monitored by in vivo 31P and 13C nuclear magnetic resonance

Cell suspensions of a respiratory deficient mutant of Saccharomyces cerevisiae were monitored by in vivo ³¹P and ¹³C Nuclear Magnetic Resonance in order to evaluate the effect of ethanol in intracellular pH and metabolism. In the absence of an added energy source, ethanol caused acidification of the cytoplasm, as indicated by the shift to higher field of the resonance assigned to the cytoplasmic orthophosphate. Under the experimental conditions used this acidification was not a consequence of an increase in the passive influx of H⁺. With cells energized with glucose, a lower value for the cytoplasmic pH was also observed, when ethanol was added. Furthermore, lower levels of phosphomonoesters were detected in the presence of ethanol, indicating that an early event in glycolysis is an important target of the ethanol action. Acetic acid was identified as responsible for the acidification of the cytoplasm, in experiments where [¹³C]ethanol was added and formation of labeled acetic acid was detected. The intracellular and the extracellular concentrations of acetic acid were respectively, 30 mM and 2 mM when 0.5% (120 mM) [¹³C]ethanol was added.Abbreviations P_i inorganic phosphate - P_ic inorganic phosphate in the cytoplasm - P_iv inorganic phosphate in the vacuole - tP terminal phosphate in polyphosphate     

Effects of vanadate on the ATP content,ATPase activity and phosphate absorption capacity of maize roots

Although the sensitivity of the plasma membrane H⁺-ATPase to vanadate is well known, the metabolic response of plant cells to vanadate is less well characterised in vivo and its use as an inhibitor in whole plant experiments has had mixed success. Experiments with maize (Zea mays, L.) roots and with purified plasma membrane fractions from the same tissues showed that exposure to vanadate caused: (i) a reduction in the capacity for phosphate uptake; (ii) a reduction in the extractable ATPase activity from the tissue; and (iii) a significant increase in the ATP level. The measurements on the extractable ATPase activity and the ATP level showed that the effect of vanadate developed slowly, apparently reflecting the slow accumulation of intracellular vanadate. The marked effect of vanadate on the ATP level-exposure to 500 M vanadate for 5 h doubled the ATP content of the roots tips-indicates that there is no stringent control over the ATP level in the roots and that the plasma membrane H⁺-ATPase activity is likely to have a significant role in determining the ATP level under normal conditions.     

Studies on the Production of Digitalis Cardenolides by Plant Tissue Culture III. Effects of Nutrients on Digitoxin Formation by Shoot-Forming Cultures of Digitalis purpurea L. Grown in Liquid Media

Shoot-forming cultures of Digitalis purpurea L. were grown invarious modifications of Murashige-Skoog medium. The effectsof vitamins, natural extracts, plant growth substances, carbonand nitrogen sources and phosphate on their growth and digitoxinformation were investigated. Sucrose, glucose and raffinosewere suitable carbon sources for both growth and digitoxin formation.The optimum concentration of sucrose was 3%. Reduction of thebasal nitrogen concentration by one-third increased the digitoxincontent per unit weight without suppression of growth. The optimalratio of nitrate-nitrogen to ammonium-nitrogen was 2, as providedin the basal medium. A threefold increase in the phosphate concentrationimproved both growth and digitoxin content. Thiamine.HCl andmyo-inositol were required for digitoxin formation, whereasglycine, nicotinic acid and pyridoxine.HCl, usual componentsof the medium, were not required for either growth or digitoxinformation during the three passages examined. Coconut milk improvedgrowth with no reduction in the digitoxin content per unit weight.Gibberellic and abscisic acids at 0.01 to 0.1 mg per liter slightlyimproved digitoxin formation. Kinetin had no clear positiveeffect on either growth or digitoxin formation. (Received March 12, 1982; Accepted August 5, 1982)     

Intracellular free zinc and zinc buffering in human red blood cells

Summary The effect of vasopressin on voltage-sensitive Ca²⁺ currents in the rat insulinoma cell line RINm5F has been investigated in patch-clamp whole-cell and single-channel current recording experiments. In the whole-cell recording configuration the dominant inward current in the presence of tetrodotoxin was noninactivating and had a high voltage threshold. This current was much enhanced when external Ca²⁺ was replaced by Ba²⁺ and was blocked by 1 m nifedipine. It can therefore be classified as an L-current. Vasopressin enhanced the L-current without changing the voltage threshold of activation or the voltage at which the peak current was observed. Vasopressin effects were seen at concentrations as low as 0.01nm, and the maximal effect was observed at about 1nm. In higher concentrations the vasopressin effects were weaker, with effects at 50nm of about the same magnitude as at 0.01nm. In single-channel current recording experiments carried out with the cell-attached configuration there were no effects on single L-channel currents when vasopressin was added to the bath solution, but in experiments in which vasopressin (5nm) was infused into the patch pipette a marked increase in the apparent channel open state probability was observed. We conclude that vasopressin, a peptide that is known to markedly enhance glucose-evoked insulin secretion, stimulates opening of the voltage-sensitive Ca²⁺ channels in insulin-secreting cells.     

Metabolic flux analysis ofBeijerinckia indica for PS-7 production

In order to investigate central metabolic changes inBeijerinckia indica, cells were grown on different carbon sources and intracellular flux distributions were studied under varying concentrations of nitrogen. Metabolic fluxes were estimated by combining material balances with extracellular substrate uptake rate, biomass formation rate, and exopolysaccharide (EPS) accumulation rate. Thirty-one metabolic reactions and 30 intracellular metabolites were considered for the flux analysis. The results revealed that most of the carbon source was directed into the Entner-Doudoroff pathway, followed by the recycling of triose-3-phosphate back to Hexose-6-phosphate. The pentose phosphate pathway was operated at a minimal level to supply the precursors for biomass formation. The different metabolic behaviors under varying nitrogen concentrations were observed with flux analysis.     

Effects of Nutritional and Hormonal Factors on the Formation of Anti-plant-viral Protein by Mirabilis jalapa L. Cells in Suspension Culture

To obtain basic information of the production of anti-plant-viral protein by Mirabilis jalapa cells in suspension culture, the cultural conditions, especially the nutritional ones, for MAP formation and cell growth were investigated. It was found MAP formation was greatly repressed by a high concentration of 2,4-D and the addition of cytokinin. The concentration of nitrogen sources, phosphate, and CaCl₂ had marked effect. Revised medium was prepared due to the corresponding optimum concentration obtained from our investigations. MAP productivity in the revised medium became twice that in the basal medium. Changes in MAP content were also examined during successive subculturing. It was recognized that MAP content increased in the early generations, then decreased slowly during subculturings.     

Selection of Cultured Tobacco Cell Strains Producing High Levels of Ubiquinone 10 by a Cell Cloning Technique

Strains producing high levels of ubiquinone 10 were isolated from tobacco cell suspension cultures by a cell cloning technique, and the UQ content of these cell strains was analyzed by high performance liquid chromatography. Three strains with high UQ yields in suspension culture were selected and used for investigations on the necessary cultural conditions for UQ production. Sucrose concentration, 2,4-D concentration and culture temperature did not have marked effects on UQ formation. These results differed from those of the original cell lines. Furthermore, the addition of yeast extract to the medium promoted cell growth in these strains remarkably but did not enhance UQ production in the cell strains. According to these investigations, UQ production was increased to 15mg/liter and the UQ level to 1890 μg/g dry wt. of cells after 11 days of incubation.     

Ammonium and urea removal by Spirulina platensis

Different concentrations either of ammonium chloride or urea were used in batch and fed-batch cultivations of Spirulina platensis to evaluate the possibility of substituting nitrate by cheaper reduced nitrogen sources in wastewaters biotreatment. The maximum nitrogen concentration able to sustain the batch growth of this microalga without inhibition was 1.7 mM in both cases. Ammonium chloride was limiting for the growth at lower concentrations, whereas inhibition took place at higher levels. This inhibition effect was less marked with urea, likely because the enzymatic hydrolysis of this compound by urease controlled the ammonia transfer into the cell. Fed-batch experiments carried out by pulse-feeding either ammonium or urea proved that the use of these compounds as nitrogen sources can sustain the long term-cultivation of S. platensis, provided that the conditions for their feeding are accurately optimized.     

Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet

Use of single nitrogen sources in nutrient media is essential to ascertaining the relative role and regulation of nitrogen assimilatory steps, and may help identify and understand highly productive media for micropropagation and adventitious shoot formation. Eight endogenous nitrogen-containing ions or compounds in sugarbeet (nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine and choline) were examined for ability to serve as sole nitrogen source for shoot or leaf disc culture of sugarbeet (Beta vulgaris L.) model clone REL-1. The most productive concentrations of nitrate, ammonium, urea, and glutamine as sole nitrogen sources were moderately supportive of shoot multiplication (64, 70, 81 and 71%, respectively) and fresh weight increase (65, 41, 54 and 41%, respectively) compared to shoot culture growth with the Murashige-Skoog nitrogen mix of 40 mM nitrate and 20 mM ammonium. Glutamate and proline were at best poorly supportive, and glycine betaine and choline were non-supportive. Callus initiation from leaf discs was supported only by nitrate, ammonium, urea, glutamine and proline (50, 100, 100, 100 and 80%, respectively, at the best concentrations, of that on Murashige-Skoog medium). Subsequent shoot regeneration from the intact disc callus in those cultures only occurred on media with nitrate, urea, glutamine, or proline (12, 3, 28 and 3% as many shoots, respectively, as on Murashige-Skoog medium). Overall, the Murashige-Skoog nitrogen mix was superior or equal to any single nitrogen source. However, single nitrogen source media with nitrate, ammonium, urea, glutamine or proline should have significant utility for shoot or leaf disc cultures of mutants with impaired nitrogen assimilation, in comparative physiology studies, or in dual cultures with pathogens of limited ability to use any of these forms of nitrogen. This revised version was published online in June 2006 with corrections to the Cover Date.     

施肥对麦田土壤可溶性有机氮的影响

利用长期定位试验,研究施肥和小麦生长对土壤可溶性有机氮(EON)的影响。长期不同施肥土壤包括不施肥(No-F)、施用化肥(NPK)和有机肥与化肥配施(MNPK)3种。EON含量范围为7.5—29.3 kg/hm~2,No-F、NPK和MNPK土壤中EON分别占可溶性总氮的40%、56%和56%。长期有机肥与化肥配施显著提高0—15 cm土层EON含量,但对30 cm以下土层EON含量无影响。在小麦开花期,可溶性有机氮的含量及其相对含量显著高于拔节期和收获期。虽然施用氮肥对当季EON含量无显著影响,但同位素示踪微区试验表明,土壤耕层(0—15 cm)中仍有0.4%—2.8%的可溶性有机氮来源于当季施入的肥料氮。可见,化学氮肥向可溶性有机氮的转化缓慢,但农田土壤中可溶性有机氮含量与矿质态氮含量相当,发生淋溶损失的风险大。     

Regulation of passive potassium transport of normal and transformed 3T3 mouse cell cultures by external calcium concentration and temperature

Summary Regulation of passive potassium ion transport by the external calcium concentration and temperature was studied on cell cultures of 3T3 mouse cells and their DNA-virus transformed derivatives. Upon lowering of external calcium concentration, passive potassium efflux generally exhibits a sharp increase at about 0.1mm. The fraction of calcium-regulated potassium efflux is largely independent of temperature in the cases of the transformed cells, but shows a sharp increase for 3T3 cells upon increasing temperature above 32°C. In the same range of temperature, the 3T3 cells exhibit the phenomenon of high-temperature inactivation of the residual potassium efflux at 1mm external calcium. At comparable cellular growth densities, the transformed cell lines do not show high-temperature inactivation of residual potassium efflux. These results are consistent with the notion of a decisive role of the internal K⁺ concentration in the cell-density dependent regulation of cell proliferation. In particular, the growth-inhibiting effect of lowering the external Ca²⁺ concentrations is considered as largely due to a rise of passive K⁺ efflux and a subsequent decrease of internal K⁺ concentration. The experimental data on the Ca²⁺ dependence of passive K⁺ flux are quantitatively described by a theoretical model based on the constant field relations including negative surface charges on the external face of the membrane, which cooperatively bind Ca²⁺ ions and may concomitantly undergo a lateral redistribution. The present evidence is consistent with acidic phospholipids as representing these negative surface charges.This work is dedicated to the memory of Max Delbrück (deceased March 10, 1981), in whose laboratory in 1966 the earlier version of the present theoretical model was developed by one of the authors.     

Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: Competition with H2PO 4 −

The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F₁ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H₂PO ₄ ^– is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15M). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theV _max, and an increase in theK _m for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H₂PO ₄ ^– is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H₂PO ₄ ^– interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F₁ remains at about 40% of the activity of the control in a concentration range of TFP of 40–100M, whereas in oxidative phosphorylation 14M TFP produces a 60% inhibition of phosphate uptake.     

Effect of phosphorus and nitrogen on growth,chlorophyll, amino nitrogen,soluble sugar contents and algal heterocysts of water fernAzolla pinnata

The P uptake inAzolla pinnata in mineral medium increased linearly with increasing phosphate doses and optimum growth and chlorophyll content of fern were recorded at 1.2 mM of K₂HPO₄. The higher doses of phosphate reduced growth and chlorophyll, but an increase in soluble sugar and amino nitrogen was noticed. Addition of combined nitrogen sources decreased the fern growth, soluble sugar, chlorophyll and heterocyst frequency but amino nitrogen was comparatively higher. The growth and chlorophyll content were always high when the fern was grown in the absence of added nitrogen sources; this supports the thesis about the specific importance of symbiosis for the nutrition of fern with nitrogen which cannot be replaced by mineral compounds.     

MPP+ toxicity in rat striatal slices: Relationship between non-selective effects and free radical production

Incubations of rat striatal slices have been used to assay MPP⁺ neurotoxicity. MPP⁺, at concentrations of 1 mM or higher, caused a marked increase in hydroxyl radicals, measured as malondialdehyde (MDA) accumulation, but not in nitric oxide production. At these doses, MPP⁺ showed an effect on dopamine terminals, causing a massive dopamine decrease, and on non-neuronal glial cells, where a marked reduction in glutamine synthetase activity was detected. At lower concentrations (25 μM), the toxic effect on dopaminergic endings was maintained without increasing malondialdehyde concentrations or inhibiting glutamine synthetase activity. The effect on glutamine synthetase was prevented by the addition to the medium of 0.5% dimethyl sulfoxide, a hydroxyl-radical scavenger, but this did not protect the effect of dopamine depletion. We propose that non-selective effects of MPP⁺, at doses of 1 mM or higher, are mediated by extracellular overproduction of hydroxyl radicals. The main factor responsible for this overproduction would not be the released dopamine but rather the MPP⁺ itself, through non selective inhibition of the mitochondrial respiratory chain or through a redox cycling that can trigger oxygen radical production.     

Nitrogen and Phosphorus in a Stretch of the Guadalupe River,Texas, with Five Main-Stream Impoundments by

Nitrogen and phosphorus were studied in a 168-km stretch of the Guadalupe River that had five main-stream impoundments. Flow through the study area was controlled by releases from these five reservoirs and from Canyon Reservoir, a deep-storage reservoir, located 30 km upstream. Parameters measured monthly on a diel basis at 16 stations were nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, Kjeldahl nitrogen, inorganic phosphate phosphorus, organic phosphate phosphorus, and total phosphate phosphorus.Inorganic nitrogen concentrations observed in this study were as high or higher than that previously reported for other bodies of water. Nitrate nitrogen entered the study area in relatively high concentrations from Comal Springs which was a major source of water for the Guadalupe River. Water from Canyon Reservoir, the other major source of water, was relatively low in nitrate nitrogen. The concentration of nitrate nitrogen was, therefore, dependent in part upon the portion of the total river flow originating from the two sources. Increased discharge from Canyon Reservoir and utilization by plants in areas of high chlorophyll a resulted in low nitrate-nitrogen levels. Retention of water in reservoirs reduced the concentration of nitrate nitrogen due to increased utilization by plants in areas of low flow. Nitrate nitrogen, in general, reached seasonal minima in summer and maxima in winter. Nitrite nitrogen showed considerable variation with no meaningful pattern except that higher concentrations occurred in association with high chlorophyll a and high Kjeldahl nitrogen, regions and periods of low river flow, and large phytoplankton populations. There was no increase in concentration of any form of nitrogen in the vicinity of sewage outfalls and no downstream accrual.Phosphorus levels in the study area were as high or higher than those reported in studies of other bodies of water. Sewage treatment plants at New Braunfels and Seguin, Texas, were major sources of phosphorus to the Guadelupe River. Total phosphate phosphorus was determined to be the most critical phosphate parameter in assessing eutrophication. Seasonally, it ranged from a winter high to a summer low. Concentrations were highest immediately below sewage outfalls and decreased as water progressed downstream. Inorganic-phosphate-phosphorus concentrations showed no clear seasonal trend but were clearly associated with sewage outfalls. Since large standing crops of phytoplankton were observed in areas of low inorganic phosphate phosphorus, it was not considered to limit photosynthesis. Total organic phosphate phosphorus varied seasonally, with high concentrations occurring during the spring and low concentrations in the fall. Total organic phosphate phosphorus showed no correlation with sewage outfalls, but was correlated to a degree with total Kjeldahl nitrogen and chlorophyll a. No consistent pattern of diel fluctuations was evident for any phosphorus or nitrogen compounds analyzed.     

Selection and Characterization of Nickel-Tolerant Tobacco Cells

Tobacco (Nicotiana tabacum L. cv. BY-2) cell lines tolerant to 700 M Ni in which unselected cells can not grow, were selected. The Ni-tolerant cells were also more tolerant to Co, but not to Cd than unselected cells. Ni concentrations in Ni-tolerant cells were always higher than those in medium. Since buthionine sulfoximine did not affect their Ni-tolerance, it is suggested that phytochelatins are not involved in Ni-tolerance of Ni-tolerant cells. On the other hand, histidine contents in Ni-tolerant and unselected cells, which were treated with Ni, were higher that those treated without Ni, and the degree of the elevation of histidine contents by Ni-treatment was higher in Ni-tolerant cells than in unselected cells. Additionally, exogenous histidine reduced the inhibitory effect of Ni on the growth of unselected cells. In addition, the cells that were tolerant to histidine-analogue, had higher contents of histidine and Ni-tolerance. These results suggest that histidine is involved in Ni-tolerance and the detoxification of Ni in symplast in Ni-tolerant cells.