Release of carboxylic anions and protons by tomato roots in response to ammonium nitrate ratio and pH in nutrient solution

The exudation of certain organic anions and protons by roots which may affect solubility of metals and P and uptake by plants, is affected by nitrogen form and pH. The objective of this work was to study exudation of carboxylates and H⁺/OH^– by tomato plants in response to NH₄/NO₃ ratio and pH in nutrient solution. Four NH₄/(NH₄+NO₃) ratios (R= 0, 0.33, 0.67 and 1) and constant vs. variable solution pH treatments were investigated. The sum of the exudation rates of all carboxylates tended to decline with increasing R, particularly tri- and dicarboxylates. The molar fraction of the exuded tri- and dicarboxylates, averaged over all treatments and plant ages, increased in the order tartarate 2%), malate (6%), succinate (15%), citrate (26%) and fumarate (46%). At R=1 the solution pH dropped from 5.2 to 3 and at R=0 increased to 8. The R corresponding to the pH stat of tomato plant was 0.3. For the constant solution pH treatment, the effect of solution pH on carboxylate exudation rate was small as compared to the effect of R. The exudation of citrate and H⁺ efflux which were initiated when NO₃ and NH₄ uptake rates per plant exceeded certain threshold values, increased with plant age.     

Coral growth in high-nutrient,low-pH seawater: a case study of corals cultured at the Waikiki Aquarium,Honolulu, Hawaii

Fifty-seven species of hermatypic corals have been maintained and grown in high-nutrient seawater at the Waikiki Aquarium, Honolulu, Hawaii. In this study we document the chemical conditions of aquarium water in terms of dissolved nutrients and carbon. Aquarium water is characterized by concentrations of inorganic nutrients that are high relative to most natural reef ecosystems: SiO₃ 200 M; PO₄ 0.6 M; NO₃ 5 M; NH₄ 2 M. In contrast, concentrations of organic nutrients are lower than most tropical surface ocean waters: DOP 0.1 M and DON 4 M. The incoming well-water servicing the facility has low pH, crating over-saturation of carbon dioxide. The coral communities in aquaria took up inorganic nutrients and released organic nutrients. Rates of nutrient uptake into aquaria coral communities were similar to nutrient uptake by natural reef communities. Coral growth rates were near the upper rates reported from the field, demonstrating corals can and do flourish in relatively high-nutrient water. The growth of corals does not appear to be inhibited at concentrations of nitrogen up to 5 M. Statements implying that corals can only grow in low nutrient oligotrophic seawater are therefore oversimplifications of processes that govern growth of these organisms. Some basic guidelines are given for maintenance of coral communities in aquaria.     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Electrical properties of parenchymal cell membranes in the oat coleoptile

Summary Parenchymal cells of oat (Avena sativa) coleoptiles had an osmotic concentration of 410 mM (determined by plasmolysis); of this only 22 mM was K⁺ and 1 mM Na⁺ (flame photometry). Cells were impaled with micropipette electrodes. Iontophoretic injection of the dye Niagara sky-blue from the micropipette showed that the tip of the electrode penetrated the vacuole. When sections of tissue were immersed in a solution of 22 mM KCl, 1 mM CaCl₂, and 50 mM glucose, average membrane potential was found to be 38.5 mV inside negative specific membrane resistance was 510 cm², and specific membrane capacitance, 2 f cm^-2. The cell membranes showed <25% retification and no electrical excitability. Electrotonic coupling of adjacent cells could not be demonstrated.     

An analysis of flash-induced electrical transients of a BLM containing chloroplast lamella extracts

Summary The electrical transients produced by chloroplast bilayer lipid membranes (Chl-BLM) from flash excitation are seen to result from three photocurrents and a discharge current. Each of the three photo-initiated charge transports in Chl-BLM (designated as Components A, B and C) exhibits an action spectrum similar to chlorophyll absorption spectra. The fast components (A and B), which are induced by electron acceptors such as Fe⁺³, have rise-times of 3 sec and 20 msec, and occur in TLM (thin lipid membranes, i.e., colored membranes up to 1 thick) as well as in BLM. Component C is induced by a transmembrane pH difference or applied voltage, has a rise-time of 1 sec, and occurs only in BLM. Component C is associated with exciton dissociation and proton transport. The mobility of the Component A current carriers in TLM is estimated to be about 1×10^–2 cm²/volt sec, and are, hence,electronic. The photovoltage waveforms are described by equations developed, which consider Component A as being caused by a direct charge separation proportional to the illumination intensity (within 0.5 sec), and Components B and C being caused by two types of exciton processes which cause charge transport after the illumination period.     

Synthesis of nanophase hydroxyapatite by a <Emphasis Type="Italic">Serratia</Emphasis> sp. from waste-water containing inorganic phosphate

Synthesis of nanophase hydroxyapatite (HA) on a bacterial surface was achieved at the expense of CaCl₂ and inorganic phosphate (Pi). After initial nucleation, calcium was precipitated on and around the cells as calcium phosphate at the expense of inorganic phosphate in the challenge solution, with no precipitation in cell-free controls. HA was also biomanufactured using inorganic phosphate ions scavenged from a phosphate-containing waste-water. With additional Ca²⁺, the concentration of phosphate was decreased from 0.27 (25ppm) to 0.02m (2ppm) in the waste-water. Crystals of calcium phosphate manufactured by the cells were located by scanning electron microscopy (SEM) and identified as HA by X-ray powder diffraction, with an average crystal size calculated as 25nm. Possible application of bioHA as a biomaterial and implications for one-step `waste-into product' are discussed.     

The association of distinct acid phosphatases with the flagella pocket and surface membrane fractions obtained from bloodstream forms of Trypanosoma rhodesiense

Summary Cell fractionation of bloodstream Trypanosoma rhodesiense, using isopycnic sucrose gradient centrifugation, reveals acid phosphatase activities against a range of substrates to be associated, to varying degrees, with subcellular particle populations identified as derived from flagella pocket membrane and surface membrane. Using these same substrates ( and glycerophosphate, p-nitrophenyl phosphate and glucose-6-phosphate) at least two distinct acid phosphatase activities can be distinguished. One is thermolabile ( 80% inactivated after 30 min. at 60°C), sensitive to tartrate (50% inhibited at 1.8 mM Na tartrate) with a pH optimum 4.5 and appears to exhibit little substrate preference. The other acid phosphatase is relatively heat stable (30% inactivated), insensitive to tartrate (> 5.0% inhibited using 1.8 mM Na tartrate) exhibits a somewhat higher pH optimum ( 6.0) and is more substrate specific (6 × more active toward glucose-6-PO₄ than -glycerophosphate). Further cell fractionation experiments reveal 85% of the tartrate sensitive acid phosphatase to be associated with flagella pocket membrane and to account for 80% of the organisms hydrolytic activity toward -glycerophosphate. The tartrate resistant acid phosphatase however, has a much less exclusive localization being almost equally distributed between surface membrane (40%) and flagella pocket membrane (60%).     

Effects of L-Glutamate Transport Inhibition by a Conformationally Restricted Glutamate Analogue (2S,1'S,2'R)-2-(Carboxycyclopropyl)Glycine (L-CCG III) on Metabolism in Brain Tissue In Vitro Analysed by NMR Spectroscopy

(2S,1'S,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG III) was a substrate of Na⁺-dependent glutamate transporters (GluT) in Xenopus laevis oocytes (IC₅₀ 13 and 2 M for, respectively, EAAT 1 and EAAT 2) and caused an apparent inhibition of [³H]L-glutamate uptake in mini-slices of guinea pig cerebral cortex (IC₅₀ 12 M). In slices (350 M) of guinea pig cerebral cortex, 5 M L-CCG III increased both the flux of label through pyruvate carboxylase and the fractional enrichment of glutamate, GABA, glutamine and lactate, but had no effect on total metabolite pool sizes. At 50 M L-CCG III decreased incorporation of ¹³C from [3-¹³C]-pyruvate into glutamate C4, glutamine C4, lactate C3 and alanine C3. The total metabolite pool sizes were also decreased with no change in the fractional enrichment. Furthermore, L-CCG III was accumulated in the tissue, probably via GluT. At lower concentration, L-CCG III would compete with L-glutamate for GluT and the changes probably reflect a compensation for the missing L-glutamate. At 50 M, intracellular L-CCG III could reach > 10 mM and metabolism might be affected directly.     

The Effect of Salicylic Acid on Peroxidase Activity in Wheat Calli Cocultured with the Bunt Pathogen Tilletia caries

The effect of salicylic acid (SA) on peroxidase activity in wheat (Triticum aestivum L.) calli cocultured with the bunt pathogen Tilletia caries was studied. Fungal infection was shown to activate cytoplasmic peroxidase. SA suppressed total peroxidase activity but did not inhibit the peroxidase with pI 9.8. A novel chitin-specific peroxidase with pI 3.5 appeared after the SA treatment. The infection of SA-treated cells with Tilletia caries activated the isoenzymes with pI 3.5, 4.8, and 7.5 and stimulated their secretion into the culture medium. The ability of SA to control wheat peroxidase activity during pathogenesis is discussed. The important role of this control in plant defense responses to the bunt pathogen is emphasized.     

Correlation between 15N NMR chemical shifts in proteins and secondary structure

Summary An empirical correlation between the peptide ¹⁵N chemical shift, ¹⁵N_i, and the backbone torsion angles _i, _i–1 is reported. By using two-dimensional shielding surfaces (_i1–1), it is possible in many cases to make reasonably accurate predictions of ¹⁵N chemical shifts for a given structure. On average, the rms error between experiment and prediction is about 3.5 ppm. Results for threonine, valine and isoleucine are worse (4.8 ppm), due presumably to ₁-distribution/-gauche effects. The rms errors for the other amino acids are 3 ppm, for a typical maximal chemical shift range of 15–20 ppm. Thus, there is a significant correlation between ¹⁵N chemical shift and secondary structure.     

New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

Pepsin fragmentation of botulinum type E neurotoxin: Isolation and characterization of 112, 48, 46, and 16 kD fragments

Controlled digestion of 150 kD single chain botulinum type E neurotoxin with pepsin atpH 6.0 produced 112, 48, 46, and 16 kD fragments. These were chromatographically purified; their locations in the 1300 amino acid residue long neurotoxin were determined by identifying the amino terminal 10 residues of 112 and 48 kD fragments, 50 residues of 46 kD fragment, and 59 residues of 16 kD fragment. The 48 and 112 kD fragments contain the N-terminal segment of the neurotoxin (i.e., residue no. 1 to 425 and 1 to 990, respectively), the 46 kD fragment corresponds to 407 residues of the C-terminal region, and the 16 kD fragment contains the 140 residues from a segment nearer to the C-terminus. The 48 kD fragment is similar to the 50 kD N-terminal light chain of the 150 kD dichain neurotoxin, which is generated by tryptic cleavage of the 150 kD single chain neurotoxin, and is separated from the 100 kD C-terminal heavy chain by dithiothreitol (DTT) reduction of an intrachain disulfide bond in the presence of 2 M urea (Sathyamoorthy and DasGupta,J. Biol. Chem. 260, 10461, 1985). The pepsin-generated 48 kD fragment, unlike the light chain, was isolated without exposure to DTT and urea. The single chain 112 kD fragment following trypsin digestion yielded 48 and 60 kD fragments that were separable after DTT reduction of the intrachain disulfide which links them. The N-terminal residues of the smaller fragment were identical to that of the single chain 150 kD neurotoxin; the single chain 112 kD fragment is therefore the neurotoxin minus the 50 kD C-terminal half of the heavy chain. The biological activities of the 48 and 112 kD fragments can be demonstrated in permeabilized PC12 cells (Lomnethet al., J. Neurochem. 57, 1413, 1991); they inhibit norepinephrine release.     

Presynaptic Modulation of K+-Evoked [3H]Dopamine Release in Striatal and Frontal Cortical Synaptosomes of Normotensive and Spontaneous-Hypertensive Rats

Regional differences in presynaptic [³H]dopamine ([³H]DA) release and its modulation by D₂ DA-receptors between the frontal cortex and striatum obtained from Wystar-Kyoto (WKY) and spontaneous-hypertensive rats (SHR) have been evaluated using superfused synaptosomes. Synaptosomal tritium content was significantly lower in the frontal cortex than in the striatum in both SHR and WKY (45% and 48%, respectively), but no differences in tritium content were obtained between strains. However, the 15 mM K⁺-evoked [³H]DA overflow was lower in the SHR as compared to WKY rats in both brain regions (striatum 23%, frontal cortex 21). Concentration-response curves for quinpirole (1nM-10 M)-mediated inhibition of 15mM K⁺-evoked [³H]DA release showed no differences between SHR and WKY. These results suggest that SHR has less ability to release [³H]DA as compared to WKY rats, but SHR did not show differences in the autoregulation of such release in both the frontal cortex and striatum.     

Subunit distribution of calcium-binding sites in Lumbricus terrestris hemoglobin

The giant, 3.6-MDa hexagonal bilayer hemoglobin (Hb) of Lumbricus terrestris consist of twelve 213-kDa globin subassemblies, each comprised of three disulfide-bonded trimers and three monomer globin chains, tethered to a central scaffolding of 36–42 linkers L1–L4 (24–32 kDa). It is known to contain 50–80 Ca and 2–4 Cu and Zn; the latter are thought to be responsible for the superoxide dismutase activity of the Hb. Total reflection X-ray fluorescence spectrometry was used to determine the Ca, Cu, and Zn contents of the Hb dissociated at pH 2.2, the globin dodecamer subassembly, and linker subunits L2 and L4. Although the dissociated Hb retained 20 Ca²⁺ and all the Cu and Zn, the globin subassembly had 0.4 to 3 Ca²⁺, depending on the method of isolation, and only traces of Cu and Zn. The linkers L2 and L4, isolated by reversed-phase high-pressure liquid chromatography at pH 2.2, had 1 Ca per mole and very little Cu and Zn. Electrospray ionization mass spectrometry of linker L3 at pH 2.2 and at neutral pH demonstrated avid binding of 1 Ca²⁺ and additional weaker binding of 7 Ca²⁺ in the presence of added Ca²⁺. Based on these and previous results which document the heterogeneous nature of the Ca²⁺-binding sites in Lumbricus Hb, we propose three classes of Ca²⁺-binding sites with affinities increasing in the following order: (i) a large number of sites (>100) with affinities lower than EDTA associated with linker L3 and dodecamer subassembly, (ii) 30 sites with affinities higher than EDTA occurring within the cysteine-rich domains of linker L3 and dodecamer subassembly, and (iii) 25 very high affinity sites associated with the linker subunits L1, L2, and L4. It is likely that the low-affinity type (i) sites are the ones involved in the effects of 1–100 mM Group IIA cations on Lumbricus Hb structure and function, namely increased stability of its quaternary structure and increased affinity and cooperativity of its oxygen binding.     

GTPγS-induced calcium transients and exocytosis in human neutrophils

Exocytosis and intracellular free calcium ([Ca²⁺]_in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5-O(3-thiotriphosphate) (GTPS) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of 40s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca²⁺]_in increases from 150 nM to 600 nM with a sigmoidal time course. The peak concentration is reached within 30 s but the main increase occurs during 3s. [Ca²⁺]_in subsequently decays within 1–2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTPS-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca²⁺]_in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types.     

Microbially-enhanced chemisorption of Ni2+ ions into biologically-synthesised hydrogen uranyl phosphate (HUP) and selective recovery of concentrated Ni2+ using citrate or chloride ion

Hydrogen uranyl phosphate (HUP) deposited enzymatically on Citrobacter N14 immobilized in polyacrylamide gel removed nickel ions from solution via intercalative ion-exchange into the HUP lattice. Using flow-through columns containing 100 mg dry weight of biomass and 200–250 mg loaded uranium column saturation and breakthrough of Ni²⁺ occurred after ca. 600 ml, with a total of 30 mg Ni²⁺ loaded per column, corresponding to a molar ratio of U:Ni of 2:1, in accordance with the identity of the material as Ni(UO₂PO₄)₂, identified previously. Ni²⁺ was selectively desorbed using 100 mM sodium citrate-citric acid buffer over 140 ml or a short pulse (5 ml) of 500 mM citrate buffer followed by a water wash, giving a total recovery volume of 80 ml, with a total citrate concentration of 30 mM in the wash solution of the latter. As an alternative eluant which gives no residual BOD NaCl (0.6 M) or seawater gave comparable recovery of Ni²⁺ to the 0.5 M citrate pulse, but with a Ni²⁺ recovery volume of 40–50 ml. The concentration ratio of Ni²⁺-deposition:desorption (vol:vol) was 3–4 fold better with chloride ion than with 100 mM citrate.     

Factors controlling long-term changes in soil pools of exchangeable basic cations and stream acid neutralizing capacity in a northern hardwood forest ecosystem

Understanding the factors regulating the concentrations of basic cations in soils and surface waters is critical if rates of recovery are to be predicted in response to decreases in acidic deposition. Using a dynamic simulation model (PnET-BGC), we evaluated the extent to which atmospheric deposition of strong acids and associated leaching by strong anions, atmospheric deposition of basic cations through changes in emissions of particulate matter, and historical forest cutting have influenced soil pools of exchangeable basic cations and the acid-base status of stream water at the Hubbard Brook Experimental Forest (HBEF) in New Hampshire. Historical deposition of basic cations was reconstructed from regression relationships with particulate matter emissions. Simulation results indicate that the combination of these factors has resulted in changes in the percent soil base saturation, and stream pH and acid neutralizing capacity (ANC) from pre-industrial estimates of 20%, 6.3 and 45 eq L^–1, respectively, to current values of 10%, 5.0 and –5 eq L^–1, respectively. These current values fall within the critical thresholds at which forest vegetation and aquatic biotic are at risk from soil and surface water acidification due to acidic deposition. While the deposition of strong acid anions had the largest impact on the acid-base status of soil and stream water, the reduction in deposition of basic cations associated with reductions in particulate emissions was estimated to have contributed about 27% of the depletion in soil Ca²⁺ exchange pool and 15% of the decreases in stream water concentrations of basic cations. Decline in stream water concentrations of basic cation occurred under both increasing and decreasing exchangeable pools, depending on the process controlling the acid base status of the ecosystem. Model calculations suggest that historical forest cutting has resulted in only slight decreases in soil pools of exchangeable basic cations, and has had a limited effect on stream ANC over the long-term.     

Conditions for high frequency embryogenesis from orange (Citrus sinensis Osb.) protoplasts

Using Trovita orange (Citrus sinensis Osb.) protoplasts isolated from 6-year-old nucellar callus, the effects of protoplast density and mannitol concentration on cell divisions and embryoid formation were examined.Somatic embryogenesis in nearly direct manner was observed only at a combination of low cell densities (4×10⁴/ml) and low mannitol concentrations (0.4 M). Two alternatives to achieve high frequency embryogenesis (70%) were to either dilute the cells to lower densities, or to do serial transfers of cells to fresh medium.Orange protoplasts (cells) showed embryogenic potential, and repression of embryogenesis occurred when protoplasts were cultured at a high density and/or under high osmotic pressure.     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

Effects of taurine on the phosphorylation of specific proteins in subcellular fractions of the rat retina

The effects of 20 mM taurine on the phosphorylation of specific proteins in mitochondrial and rod outer segment subcellular fractions of the rat retina were measured. A band of protein with an apparent molecular wieght of 20K was consistently inhibited by taurine. Densitometry measurements performed on gel electrophoresis autoradiograms from the mitochondrial fraction demonstrated a 42.7±8.3% decrease due to taurine (20 mM) in the area corresponding to radioactivity from the 20K phosphoprotein. However, only a 21.2±9.0% decrease was observed due to taurine in the rod outer segment preparation. These data suggest that taurine is exerting its primary effect on the phosphorylation of the 20K molecular weight protein in the mitochondria of the retina. In addition, calmodulin and phorbol ester had no effect on the phosphorylation of the 20K molecular weight protein.