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At least 55 records · Page 3

Polyamine binding to proteins in oat and Petunia protoplasts

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.

NASA Discipline Plant Biology↗

Cyclic variations in nitrogen uptake rate of soybean plants: ammonium as a nitrogen source

When NO3- is the sole nitrogen source in flowing solution culture, the net rate of nitrogen uptake by nonnodulated soybean (Glycine max L. Merr. cv Ransom) plants cycles between maxima and minima with a periodicity of oscillation that corresponds with the interval of leaf emergence. Since soybean plants accumulate similar quantities of nitrogen when either NH4+ or NO3- is the sole source in solution culture controlled at pH 6.0, an experiment was conducted to determine if the oscillations in net rate of nitrogen uptake also occur when NH4+ is the nitrogen source. During a 21-day period of vegetative development, net uptake of NH4+ was measured daily by ion chromatography as depletion of NH4+ from a replenished nutrient solution containing 1.0 millimolar NH4+. The net rate of NH4+ uptake oscillated with a periodicity that was similar to the interval of leaf emergence. Instances of negative net rates of uptake indicate that the transition between maxima and minima involved changes in influx and efflux components of net NH4+ uptake.

NASA Discipline Life Support Systems↗

Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

NASA Discipline Number 29-20↗

Somatic proembryo production from excised, wounded zygotic carrot embryos on hormone-free medium: evaluation of the effects of pH, ethylene and activated charcoal

Wounded zygotic embryos of cultivated carrot produce somatic proembryos on hormone-free nutrient medium containing 1 mM NH4+ as the sole nitrogen source. Continued maintenance of proembryos on this medium leads to a "pure" culture of preglobular stage proembryos (PGSPs). Ethylene had no effect on this process. Also, somatic embryo production was not affected by growing cultures on activated charcoal-impregnated filter papers. However, somatic proembyros initiated on activated charcoal papers were not maintainable as PGSPs and developed into later embryo stages. Normally, medium pH dropped from 5.7 to 4 during each subculture period, but when using activated charcoal papers the pH endpoint was around 6 - 7 due to a leachable substance(s) within the filter papers. When powdered, activated charcoal was used in the medium as an adsorbent of products potentially released after wounding, pH dropped at the normal rate and to the expected levels; proembryos did not mature into later embryo stages and were maintainable exclusively as PGSPs. Low pH (approximately 4) is detrimental to proembyro production, but is essential to maintaining PGSPs on hormone-free nutrient medium, whereas a sustained pH > or = 5.7 allows continued development of PGSPs into later embryo stages.

NASA Program Space Biology↗

Potato growth and yield using nutrient film technique (NFT)

Potato plants, cvs Denali and Norland, were grown in polyvinyl chloride (PVC) trays using a continuous flowing nutrient film technique (NFT) to study tuber yield for NASA's Controlled Ecological Life Support Systems (CELSS) program. Nutrient solution pH was controlled automatically using 0.39M (2.5% (v/v) nitric acid (HNO3), while water and nutrients were replenished manually each day and twice each week, respectively. Plants were spaced either one or two per tray, allotting 0.2 or 0.4 m2 per plant. All plants were harvested after 112 days. Denali plants yielded 2850 and 2800 g tuber fresh weight from the one- and two-plant trays, respectively, while Norland plants yielded 1800 and 2400 g tuber fresh weight from the one- and two-plant trays. Many tubers of both cultivars showed injury to the periderm tissue, possibly caused by salt accumulation from the nutrient solution on the surface. Total system water usage throughout the study for all the plants equaled 709 liters (L), or approximately 2 L m-2 d-1. Total system acid usage throughout the study (for nutrient solution pH control) equaled 6.60 L, or 18.4 ml m-2 d-1 (7.2 mmol m-2 d-1). The results demonstrate that continuous flowing nutrient film technique can be used for tuber production with acceptable yields for the CELSS program.

NASA Discipline Number 61-20↗

Galactose inhibits auxin-induced growth of Avena coleoptiles by two mechanisms

Galactose inhibits auxin-induced growth of Avena coleoptiles by at least two mechanisms. First, it inhibits auxin-induced H(+)-excretion needed for the initiation of rapid elongation. Galactose cannot be doing so by directly interfering with the ATPase since fusicoccin-induced H(+)-excretion is not affected. Secondly, galactose inhibits long-term auxin-induced growth, even in an acidic (pH 4.5) solution. This may be due to an inhibition of cell wall synthesis. However, galactose does not reduce the capacity of walls to be loosened by H+, given exogenously or excreted in response to fusicoccin.

NASA Discipline Plant Biology↗

Determination of ammonium ion by fluorometry or spectrophotometry after on-line derivatization with o-phthalaldehyde

A fast, sensitive, simple, and highly reproducible method for routine assay of ammonium ion (NH4+) was developed by using HPLC equipment. The method is based on the reaction of NH4+ with o-phthalaldehyde (OPA) in the presence of 2-mercaptoethanol. After an on-line derivatization, the resulting NH4(+)-OPA product was quantified by using fluorometric or spectrophotometric detection. For fluorometric detection, the excitation and emission wavelengths were 410 and 470 nm, respectively. The spectrophotometric detection was made by measuring absorbance at 410 nm. Results on the effects of OPA-reagent composition and pH, reaction temperature, sample matrix, and linearity of the assay are presented. Even though it took about 2 min from the time of sample injection to the appearance of sample peak, sample injections could be overlapped at an interval of about 1 min. Thus, the actual time needed for analysis was about 1 min per assay. The method can be used in a fully automated mode by using an autosampler injector.

NASA Discipline Life Support Systems↗

NMR studies on Na+ transport in Synechococcus PCC 6311

The freshwater cyanobacterium Synechococcus PCC 6311 is able to adapt to grow after sudden exposure to salt (NaCl) stress. We have investigated the mechanism of Na+ transport in these cells during adaptation to high salinity. Na+ influx under dark aerobic conditions occurred independently of delta pH or delta psi across the cytoplasmic membrane, ATPase activity, and respiratory electron transport. These findings are consistent with the existence of Na+/monovalent anion cotransport or simultaneous Na+/H+ +anion/OH- exchange. Na+ influx was dependent on Cl-, Br-, NO3-, or NO2-. No Na+ uptake occurred after addition of NaI, NaHCO3, or Na2SO4. Na+ extrusion was absolutely dependent on delta pH and on an ATPase activity and/or on respiratory electron transport. This indicates that Na+ extrusion via Na+/H+ exchange is driven by primary H+ pumps in the cytoplasmic membrane. Cells grown for 4 days in 0.5 m NaCl medium, "salt-grown cells," differ from control cells by a lower maximum velocity of Na+ influx and by lower steady-state ratios of [Na+]in/[Na+]out. These results indicate that cells grown in high-salt medium increase their capacity to extrude Na+. During salt adaptation Na+ extrusion driven by respiratory electron transport increased from about 15 to 50%.

Non-NASA Center↗

Prebiotic synthesis of histidyl-histidine

Histidyl-histidine (His-His) has been synthesized in a yield of up to 14.4% under plausible prebiotic conditions using histidine (His), cyanamide, and 4-amino-5-imidazole carboxamide. A trace amount of His trimer was also detected. Because the imidazole group of His is involved in a number of important enzymatic reactions, and His-His has been shown to catalyze the prebiotic synthesis of glycyl-glycine, we expect this work will stimulate further studies on the catalytic activities of simple His-containing peptides in prebiotic reactions.

NASA Discipline Exobiology↗

Tomato responses to ammonium and nitrate nutrition under controlled root-zone pH

Tomato (Lycopersicon esculentum L. Mill. 'Vendor') plants were grown for 21 days in flowing solution culture with N supplied as either 1.0 mM NO3- or 1.0 mM NH4+. Acidity in the solutions was automatically maintained at pH 6.0. Accumulation and distribution of dry matter and total N and net photosynthetic rate were not affected by source of N. Thus, when rhizosphere acidity was controlled at pH 6.0 during uptake, either NO3- or NH4+ can be used efficiently by tomato. Uptake of K+ and Ca2+ were not altered by N source, but uptake of Mg2+ was reduced in NH4(+)-fed plants. This indicates that uptake of Mg2+ was regulated at least partially by ionic balance within the plant.

NASA Program CELSS↗

An evaluation of MES (2(N-Morpholino)ethanesulfonic acid) and Amberlite IRC-50 as pH buffers for nutrient solution studies

All buffering agents used to stabilize pH in hydroponic research have disadvantages. Inorganic buffers are absorbed and may become phytotoxic. Solid carbonate salts temporarily mitigate decreasing pH but provide almost no protection against increasing pH, and they alter nutrient absorption. Exchange resins are more effective, but we find that they remove magnesium and manganese from solution. We have tested 2(N-Morpholino)ethanesulfonic acid (MES) as a buffering agent at concentrations of 1 and 10 mol m-3 (1 and 10 mM) with beans, corn, lettuce, tomatoes, and wheat. MES appears to be biologically inert and does not interact significantly with other solution ions. Relative growth rates among controls and MES treatments were nearly identical for each species during the trial period. The pH was stabilized by 1 mol m-3 MES. This buffer warrants further consideration in nutrient research.

Alkanesulfonic Acids/chemistry/pharmacology↗

Utilization of ammonium as a nitrogen source: effects of ambient acidity on growth and nitrogen accumulation by soybean

Dry matter accumulation of plants utilizing NH4+ as the sole nitrogen source generally is less than that of plants receiving NO3- unless acidity of the root-zone is controlled at a pH of about 6.0. To test the hypothesis that the reduction in growth is a consequence of nitrogen stress within the plant in response to effects of increased acidity during uptake of NH4+ by roots, nonnodulated soybean plants (Glycine max [L.] Merr. cv Ransom) were grown for 24 days in flowing nutrient culture containing 1.0 millimolar NH4+ as the nitrogen source. Acidities of the culture solutions were controlled at pH 6.1, 5.1, and 4.1 +/- 0.1 by automatic additions of 0.01 N H2SO4 or Ca(OH)2. Plants were sampled at intervals of 3 to 4 days for determination of dry matter and nitrogen accumulation. Rates of NH4+ uptake per gram root dry weight were calculated from these data. Net CO2 exchange rates per unit leaf area were measured on attached leaves by infrared gas analysis. When acidity of the culture solution was increased from pH 6.1 to 5.1, dry matter and nitrogen accumulation were reduced by about 40% within 14 days. Net CO2 exchange rates per unit leaf area, however, were not affected, and the decreased growth was associated with a reduction in rates of appearance and expansion of new leaves. The uptake rates of NH4+ per gram root were about 25% lower throughout the 24 days at pH 5.1 than at 6.1. A further increase in solution acidity from pH 5.1 to 4.1 resulted in cessation of net dry matter production and appearance of new leaves within 10 days. Net CO2 exchange rates per unit leaf area declined rapidly until all viable leaves had abscised by 18 days. Uptake rates of NH4+, which were initially about 50% lower at pH 4.1 than at 6.1 continued to decline with time of exposure until net uptake ceased at 10 days. Since these responses also are characteristic of the sequence of responses that occur during onset and progression of a nitrogen stress, they corroborate our hypothesis.

NASA Discipline Number 61-10↗

Root-zone acidity affects relative uptake of nitrate and ammonium from mixed nitrogen sources

Soybean plants (Glycine max [L.] Merr. cv Ransom) were grown for 21 days on 4 sources of N (1.0 mM NO3-, 0.67 mM NO3- plus 0.33 mM NH4+, 0.33 mM NO3- plus 0.67 mM NH4+, and 1.0 mM NH4+) in hydroponic culture with the acidity of the nutrient solution controlled at pH 6.0, 5.5, 5.0, and 4.5. Dry matter and total N accumulation of the plants was not significantly affected by N-source at any of the pH levels except for decreases in these parameters in plants supplied solely with NH4+ at pH 4.5. Shoot-to-root ratios increased in plants which had an increased proportion [correction of proporiton] of NH4(+)-N in their nutrient solutions at all levels of root-zone pH. Uptake of NO3- and NH4+ was monitored daily by ion chromatography as depletion of these ions from the replenished hydroponic solutions. At all pH levels the proportion of either ion that was absorbed increased as the ratio of that ion increased in the nutrient solution. In plants which were supplied with sources of NO3- plus NH4+, NH4+ was absorbed at a ratio of 2:1 over NO3- at pH 6.0. As the pH of the root-zone declined, however, NH4+ uptake decreased and NO3- uptake increased. Thus, the NH4+ to NO3- uptake ratio declined with decreases in root-zone pH. The data indicate a negative effect of declining root-zone pH on NH4+ uptake and supports a hypothesis that the inhibition of growth of plants dependent on NH4(+)-N at low pH is due to a decline in NH4+ uptake and a consequential limitation of growth by N stress.

NASA Discipline Life Support Systems↗

Effects of root-zone acidity on utilization of nitrate and ammonium in tobacco plants

Tobacco (Nicotiana tabacum L., cv. 'Coker 319') plants were grown for 28 days in flowing nutrient culture containing either 1.0 mM NO3- or 1.0 mM NH4+ as the nitrogen source in a complete nutrient solution. Acidities of the solutions were controlled at pH 6.0 or 4.0 for each nitrogen source. Plants were sampled at intervals of 6 to 8 days for determination of dry matter and nitrogen accumulation. Specific rates of NO3- or NH4+ uptake (rate of uptake per unit root mass) were calculated from these data. Net photosynthetic rates per unit leaf area were measured on attached leaves by infrared gas analysis. When NO3- [correction of NO-] was the sole nitrogen source, root growth and nitrogen uptake rate were unaffected by pH of the solution, and photosynthetic activity of leaves and accumulation of dry matter and nitrogen in the whole plant were similar. When NH4+ was the nitrogen source, photosynthetic rate of leaves and accumulation of dry matter and nitrogen in the whole plant were not statistically different from NO3(-) -fed plants when acidity of the solution was controlled at pH 6.0. When acidity for NH4(+) -fed plants was increased to pH 4.0, however, specific rate of NH4+ uptake decreased by about 50% within the first 6 days of treatment. The effect of acidity on root function was associated with a decreased rate of accumulation of nitrogen in shoots that was accompanied by a rapid cessation of leaf development between days 6 and 13. The decline in leaf growth rate of NH4(+) -fed plants at pH 4.0 was followed by reductions in photosynthetic rate per unit leaf area. These responses of NH4(+) -fed plants to increased root-zone acidity are characteristic of the sequence of responses that occur during onset of nitrogen stress.

NASA Discipline Life Support Systems↗

Permeability of lipid bilayers to amino acids and phosphate

Permeability coefficients for amino acid classes, including neutral, polar, hydrophobic, and charged species, were measured and compared with values for other ionic solutes such as phosphate. The rates of efflux of glycine, lysine, phenylalanine, serine and tryptophan were determined after they were passively entrapped in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (EPC) or dimyristoylphosphatidylcholine (DMPC). The following permeability coefficients were obtained for: glycine, 5.7 x 10(-12) cm s-1 (EPC), 2.0 x 10(-11) cm s-1 (DMPC); serine, 5.5 x 10(-12) cm s-1 (EPC), 1.6 x 10(-11) cm s-1 (DMPC); lysine, 5.1 x 10(-12) cm s-1 (EPC), 1.9 x 10(-11) cm s-1 (DMPC); tryptophan, 4.1 x 10(-10) cm s-1 (EPC); and phenylalanine, 2.5 x 10(-10) cm s-1 (EPC). Decreasing lipid chain length increased permeability slightly, while variations in pH had only minor effects on the permeability coefficients of the amino acids tested. Phosphate permeability was in the range of 10(-12)-10(-13) cm s-1 depending on the pH of the medium. The values for the polar and charged amino acids were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium, which are in the range of 10(-12)-10(-13) cm s-1, depending on conditions and the lipid species used. This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. The results are relevant to the permeation of certain peptides into lipid bilayers during protein translocation and membrane biogenesis.

NASA Discipline Exobiology↗

Pathways of proton release in the bacteriorhodopsin photocycle

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.

NASA Discipline Exobiology↗

Muscle fatigue in frog semitendinosus: role of intracellular pH

The purpose of this study was to utilize glass microelectrodes to characterize the intracellular pH (pHi) before and during recovery from fatigue in the frog semitendinosus (ST) muscle. A second objective was to evaluate the relationship between pHi and contractile function. The frog ST muscle (22 degrees C) was fatigued by direct electrical stimulation with 100-ms 150-Hz trains at 1/s for 5 min. Peak tetanic force (Po) was reduced to 8.5% of initial force and recovered in a biphasic manner, returning to the resting value by 40 min. Resting pHi was 7.00 +/- 0.02 (n = 37) and declined with fatigue to an average value of 6.42 at 3 min of recovery. During recovery pHi significantly increased and by 25 min had returned to the prefatigue value. The pHi recovery was highly correlated to the slow phase of Po recovery (r = 0.98, P less than 0.001). The mean resting membrane potential was -78 +/- 1.0 mV (n = 42) and at 3 min of recovery was depolarized to -67 +/- 4 mV. Both the peak rate of twitch force development (+dP/dt) (r = 0.99, P less than 0.001) and decline (-dP/dt) (r = 0.94, P less than 0.014) were highly correlated to pHi during the slow phase of recovery. Contraction time (CT) and one-half relaxation time (1/2RT) increased significantly and recovered exponentially. The recovery of CT and 1/2RT were both significantly correlated to pHi (r = -0.93, P less than 0.001 and r = -0.86, P less than 0.001 for CT and 1/2RT, respectively).(ABSTRACT TRUNCATED AT 250 WORDS).

NASA Discipline Musculoskeletal↗

Physicochemical action of potassium-magnesium citrate in nephrolithiasis

Effect of potassium-magnesium citrate on urinary biochemistry and crystallization of stone-forming salts was compared with that of potassium citrate at same dose of potassium in five normal subjects and five patients with calcium nephrolithiasis. Compared to the placebo phase, urinary pH rose significantly from 6.06 +/- 0.27 to 6.48 +/- 0.36 (mean +/- SD, p less than 0.0167) during treatment with potassium citrate (50 mEq/day for 7 days) and to 6.68 +/- 0.31 during therapy with potassium-magnesium citrate (containing 49 mEq K, 24.5 mEq Mg, and 73.5 mEq citrate per day). Urinary pH was significantly higher during potassium-magnesium citrate than during potassium citrate therapy. Thus, the amount of undissociated uric acid declined from 118 +/- 61 mg/day during the placebo phase to 68 +/- 54 mg/day during potassium citrate treatment and, more prominently, to 41 +/- 46 mg/day during potassium-magnesium citrate therapy. Urinary magnesium rose significantly from 102 +/- 25 to 146 +/- 37 mg/day during potassium-magnesium citrate therapy but not during potassium citrate therapy. Urinary citrate rose more prominently during potassium-magnesium citrate therapy (to 1027 +/- 478 mg/day from 638 +/- 252 mg/day) than during potassium citrate treatment (to 932 +/- 297 mg/day). Consequently, urinary saturation (activity product) of calcium oxalate declined significantly (from 1.49 x 10(-8) to 1.03 x 10(-8) M2) during potassium-magnesium citrate therapy and marginally (to 1.14 x 10(-8) M2) during potassium citrate therapy.(ABSTRACT TRUNCATED AT 250 WORDS).

Non-NASA Center↗