Patterns in the release of gaseous ammonia by terrestrial isopods

Summary In the fall and in early spring P. scaber and O. asellus released gaseous ammonia in the form of more or less regularly spaced bursts. In the spring about twice as much ammonia was released by O. asellus than in the fall. In late spring and summer, however, both species released ammonia in a rhythmic fashion, with a maximum at noon and early in the afternoon, and a minimum early at night. Sometimes a second maximum occurred late at night.In O. asellus the addition of a moist substrate to the reaction chamber shifted the maximum of the release of ammonia from noon to late night and early morning.Fed specimens of P. scaber released only about one-third as much NH₃ as fasting animals and—at least in constant darkness—with a period of much reduced amplitude.It is concluded that the rhythmical release of ammonia is inversely related to the pattern of locomotory activity of these animals. This would implicate mechanisms that regulate either the production or the release of ammonia in such a way that the maximum occurs at a time when the animals' production of energy is at a minimum and when they are protected against loss of water by sitting in their moist retreats.The work at Innsbruck was supported by the Fonds zur Förderung der wissenschaftlichen Forschung of Austria.     

Significance of gaseous NO for ammonia oxidation by Nitrosomonas eutropha

Nitrification by the obligately lithoautotrophic ammonia oxidizer Nitrosomonas eutropha was significantly inhibited when nitric oxide was removed from the culture medium by means of intensive aeration and turbulence. Nearly complete recovery of ammonia oxidation could be achieved by adding 100 ppm NO to the supplied air. Inhibition of ammonia oxidation occurred also upon addition of the NO binding agens 2,3-Dimercapto-1-propane-sulfonic acid (DMPS). Recovery of ammonia oxidation occurred within 3 h in the presence of 100 ppm NO and within 76 h in the absence of externally added NO. In co-cultures of N. eutropha and the NO detoxifying bacterium Pseudomonas PS88, hardly any nitrification was detectable and release of NO was extremely low when the heterotroph was provided with an organic substrate. When cells of Pseudomonas PS88 were added to a mixotrophically nitrifying culture of N. eutropha the release of NO decreased drastically upon the addition and ammonia oxidation ceased. These results confirm for the first time the significance of NO in the course of ammonia oxidation by N. eutropha.     

Ion movement through gramicidin A channels. Interfacial polarization effects on single-channel current measurements.

Gramicidin A single-channel current-voltage characteristics were studied at low permeant ion concentrations and very high applied potentials. The purpose of these experiments was to elucidate the basis for the small, but definite, voltage dependence observed under these circumstances. It was found that this residual voltage dependence is a reflection of interfacial polarization effects, similar to those proposed by Walz et al. (Biophys. J. 9:1150-1159). It will be concluded that there exists an effectively voltage-independent step in the association reaction between a gramicidin A channel and the permeating ion. Some consequences of interfacial polarization effects for the analysis of conductance vs. activity relations will be discussed.     

Biocatalysis in multi-phase reaction mixtures containing organic liquids

A wide range of enzymes and whole microbial cells will act as catalysts in reaction mixtures that contain 2 or more phases, one of which is an organic liquid (either a reactant or including water-immiscible organic solvents). These "biphasic" systems have a variety of structures, knowledge of which aids predictions about biocatalyst activity and stability. There is often a dilute aqueous solution phase (containing the biocatalyst), which may be emulsified with the organic phase, or "trapped" within catalyst particles; sometimes however there may only be traces of water adsorbed to the enzyme or cells. These reaction systems offer several advantages for industrial applications, notably the higher solubilities of many reactants of interest, and the ability of readily available hydrolytic enzymes to catalyse syntheses. The most non-polar organic liquids are least likely to inactivate biocatalysts, though many do remain active with relatively polar solvents. Modification of the biocatalyst may stabilise against inactivation, especially where this is due to direct contact with the phase interface. The mass transfer processes required in these systems remain poorly understood, particularly because the interfacial area is often unknown. Attractive continuous reactors may be operated using a packed bed of catalyst with a trapped aqueous phase.     

Design of laboratory continuous-culture equipment for accurate gaseous metabolism measurements

The accuracy of kinetic and stoichiometric data obtained from most laboratory-scale continuous-culture equipment, particularly involving gaseous measurements, may be much lower than many workers realize, despite the use of good quality instruments. For example, errors in specific oxygen uptake measurements (QO(2)) easily can be as high as +/-100%. This article assesses the accuracies of individual instruments and of the overall system in greater detail than has previously been reported and suggestions are made as to how the errors can be reduced to acceptable levels.     

Biomanufacturing by in vitro biosystems containing complex enzyme mixtures

Microbial fermentation is the current predominant biomanufacturing platform. However, it suffers from low production yields, slow reaction rates, and scaling-up challenges. In vitro enzymatic biosystems are emerging to expand the traditional biotechnological mode by utilizing more than three enzymes for manufacturing the desired product from cheap substrate. In the past few years, numerous proofs of the concept of in vitro biosystems containing complex enzyme mixtures from different groups worldwide have inspired the development of these platforms for biomanufacturing, these biosystems show advantages such as near-theoretical product yields, faster reaction rates, reduced interference from toxic compounds, and unprecedented level of engineering. In this review, several examples of in vitro systems are presented to illustrate these advantages and possible solutions to overcome the remaining challenges are discussed. The continuing decrease in enzyme cost and improvements in enzyme engineering techniques will make in vitro biosystems a comparable biomanufacturing platform for microbial fermentation in the near future.     

Assimilation of gaseous ammonia and the transport of its products in barley and spinach leaves

The interactions between the assimilation and transport of nitrogenand carbon were investigated in barley and spinach leaves. Bothplants were fumigated with NH₃ (1 mg m^–3 and the contentof amino acids, sucrose and carbon intermediates of amino acidmetabolism were analysed in the leaves, apoplast and phloemsap. The following changes took place in the C- and N-metabolismof barley leaves during 5 h of fumigation with NH₃ (a) The contentsof amino acids, especially glutamine, largely increased andthe contents of sucrose, 2-oxoglutarate, phosphoenolpyruvate,and glycerate-3-phosphate declined. (b) A decrease in the phophoenolpyruvatecontent was accompanied by an increased activity of phosphoenolpyruvatecarboxylase. (c) The altered cytosolic concentrations of aminoacids and sucrose during NH₃ fumigation correlated with similarchanges in the apoplast and phloem sap. The altered percentageof each amino acid relative to the total amino acid concentrationin the cytosol, caused by NH₃ fumigation, is reflected in theapoplast and the phloem sap. The results indicate that the concentrations of amino acids in the cytosol determine their concentrationsin the phloem. Key words: Amino acids, ammonia fumigation, barley leaves, C: N partitioning, phosphoenolpyruvate carboxylase, phloem sap, spinach leaves     

Osmotic pressure measurements of ovalbumin and lysozyme mixtures

Ovalbumin and lysozyme have been reported to undergo a mixed association in solutions of low ionic strength. Osmotic pressure experiments were performed on ovalbumin and on lysozyme solutions in 0.06 M cacodylate buffer (I = 0.02, pH = 5.8) at 30 and at 37 degrees C. The individual proteins did not undergo any self-associations at either temperature; these measurements indicated that each of the solutions was nonideal. Osmotic pressure experiments on three blends of lysozyme and ovalbumin at 30 and 37 degrees C could be interpreted in two ways. One interpretation was that a nonideal, nonassociating mixture of A and B was present; for the three solutions the mixed nonideal term BAB was negative. A negative nonideal term is usually interpreted as indicating an association. The other interpretation of the data was as a quasi-ideal mixed association of the type A + B in equilibrium AB.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Acquisition and assimilation of gaseous ammonia as revealed by intracellular pH changes in leaves of higher plants

Atmospheric ammonia (NH₃) from various anthropogenic sources has become a serious problem for natural vegetation. Ammonia not only causes changes in plant nitrogen metabolism, but also affects the acid-base balance of plants. Using the pH-sensitive fluorescent dyes pyranine and esculin, cytosolic and vacuolar pH changes were measured in leaves of C₃ and C₄ plants exposed for brief periods to concentrations of NH₃ in air ranging from 1.33 to 8.29 mol NH₃ · mol^-1 gas (0.94–5.86 mg · m^-3). After a lag phase, uptake of NH₃ from air at a rate of 200 nmol NH₃ · m ^{- 2} leaf area · s^{- 1} into leaves of Zea mays L. increased pyranine fluorescence indicating cytosolic alkalinisation. The increase was much larger in the dark than in the light. In illuminated leaves of the C₃ plant Pelargonium zonale L. and the C₄ plants Z. mays and Amaranthus caudatus L., NH₃-dependent cytosolic alkalinisation was particularly pronounced when CO₂ was supplied at very low levels (16 or 20 mol CO₂ · mol^{- 1} gas, containing 210 mmol O₂ · mol^{- 1} gas). An increase in esculin fluorescence, which was smaller than that of pyranine, was indicative of trapping of some of the NH₃ in the vacuoles of leaves of Spinacia oleracea L. and Z. mays. Photosynthesis and transpiration remained unchanged during exposure of illuminated leaves to NH₃, yielding an influx of 200 nmol NH₃ · m^-2 leaf area · s^-1 for up to 30 min, the longest exposure time used. Both CO₂ and O₂ influenced the extent of cytosolic alkalinisation. At 500 mol CO₂ · mol^-1 gas the cytosolic alkalinisation was suppressed more than at 16 or 20 mol CO₂ · mol^-1 gas. The suppressing effect of CO₂ on the NH₃induced alkalinisation was larger in illuminated leaves of the C₄ plants Z. mays and A. caudatus than in leaves of the C₃ plant P. zonale. A reduction of the O₂ concentration from 210 to 10 mmol O₂ · mol ^-1 gas, which inhibits photorespiration, increased the NH₃induced cytosolic alkalinisation in C₃ plants. Suppression by CO₂ or O₂ of the alkaline pH shift caused by the dissolution and protonation of NH₃ in queous leaf compartments, and possibly by the production of organic compounds synthesised from atmospheric NH₃, indicates that NH₃ which enters leaves is rapidly assimilated if photosynthesis or photorespiration provide nitrogen acceptor molecules.This work was supported by the Biotechnology and Biological Sciences Research Council and the Deutsche Forschungsgemein-schaft within the framework of the research of Sonderforschun-gsbreich 251 of the University of Würzburg. We are grateful to Dr. B. Wollenweber (The Royal Veterinary and Agricultural University, Denmark) for discussions.     

Preservation of high-moisture maize — A comparison of gaseous and liquid anhydrous ammonia with methylene-bis-propionate

Freshly harvested maize (22–24% moisture) was treated with either gaseous ammonia (0.9% dry basis), liquid anhydrous ammonia (1.3% dry basis), or methylene-bis-propionate (0.8% dry basis) and stored in metal bins. All treatments initially decreased the counts of bacteria and moulds. Growth of these microorganisms was prevented throughout the 6-month storage period by methylene-bis-propionate and for 5 months by gaseous anhydrous ammonia. Liquid anhydrous ammonia prevented mould growth for 4 months, while bacterial counts increased to pretreatment numbers after 1 month but did not increase further until after 5 months' storage. In feeding trials with steers, more efficient weight gains were obtained from the ammonia-preserved maize than from the methylene-bis-propionate preserved maize.     

Membrane current and noise measurements in voltage-clamped node ofRanvier

Summary In voltage-clamp configurations for nodes ofRanvier the axoplasm resistance functions as a voltage-current converter. In existing configurations this resistance cannot be measured directly. In the present arrangement the electrical resistances of the preparation (axoplasm, membrane and seals) can be measured only from two measurements. This allows us to: 1. calibrate the ionic current under voltage-clamp conditions, and 2. calculate the intensity of the current fluctuations, not arising from the membrane (background noise). The measured axoplasm resistances are considerably higher than the values calculated on the basis of fiber geometry and axoplasm resistivity. The difference is due to the presence of constrictions in the nerve fiber. Membrane current estimation based on geometrical parameters in the presence of wide seals may contain large errors. Variations in the axoplasm resistance for voltage-membrane current conversion were observed within 1.5 hr. In 68% of the fibers this resistance decreased with 30% of the original value. With our current calibration the values for the maximum sodium conductance (at 0 mV membrane potential), maximum potassium conductance and leakage conductance are 49.5×10^–8, 6.66×10^–8 and 1.71×10^–8 S, respectively. The contribution of the different noise sources to the total background noise was calculated at the holding potential. For frequencies below 10³ Hz there is an excellent agreement between measured and calculated noise levels.