Metabolism of trans-Aconitic Acid in Maize : II. Regulatory Properties of Two Compartmented Forms of Citrate Dehydrase

Kinetics of two molecular forms of K-dependent citrate dehydrase in maize (Zea mays L.) are reported. The isozymes, designated CD I and CD II, were found to be compartmented in mitochondria and cytosol, respectively.

CD I exhibited hyperbolic kinetics with respect to both citrate and potassium with K_m 2.3 and 12 millimolar, respectively. Maximum velocity was 0.38 micromole of trans-aconitic acid per minute per milligram protein. The pH optimum was 7.2. trans-aconitic synthesis by CD I is regulated by both citrate concentration and pH.

CD II exhibited hyperbolic kinetics with respect to citrate (K_m 0.6 millimolar) but sigmoidal kinetics with respect to potassium. trans-aconitic acid synthesis by CD II is regulated by potassium. This may account for the positive correlation between leaf potassium and trans-aconitic acid in certain grasses (Clark 1968 Crop Sci 8: 165).

     

Analysis of Proportions in One- or Two-Way Tables with Spoiled Data

This paper discusses the analysis of data on proportions in contingency tables. The X² ‘index of dispersion test’ (e.g. Fisher, 1954) is developed in these situations and compared with the use of the ‘logit’ transformation. An example using Osborn's (1979) data is given, illustrating the estimation of one or more missing observations.     

Are there infinitely many trucks in the technosphere,or exactly one? How independent sampling of instances of unit processes affects uncertainty analysis in LCA

Purpose

Product systems use the same unit process models to represent distinct but similar activities. This notably applies to activities in cyclic dependency relationships (or “feedback loops”) that are required an infinite number of times in a product system. The study aims to test the sensitivity of uncertainty results on the assumption made concerning these different instances of the same activities. The default assumption assumes homogeneous production, and the same parameter values are sampled for all instances (e.g., there is one truck). The alternative assumption is that every instance is distinct, and parameter values are independently sampled for different instances of unit processes (e.g., there are infinitely many trucks). Intuitively, sampling the same values for each instance of a unit process should result in more uncertain results.

Methods

The results of uncertainty analyses carried out under either assumption are compared. To simulate models where each instance of a unit process is independent, we convert network models to acyclic LCI models (tree models). This is done three times: (1) for a very simple product system, to explain the methodology; (2) for a sample product system from the ecoinvent database, for illustrative purposes; and (3) for thousands of product systems from ecoinvent databases.

Results and discussion

The uncertainty of network models is indeed greater than that of corresponding tree models. This is shown mathematically for the analytical approximation method to uncertainty propagation and is observed for Monte Carlo simulations with very large numbers of iterations. However, the magnitude of the difference in indicators of dispersion is, for the ecoinvent product systems, often less than a factor of 1.5. In few extreme cases, indicators of dispersion are different by a factor of 4. Monte Carlo simulations with smaller numbers of iterations sometimes give the opposite result.

Conclusions

Given the small magnitude of the difference, we believe that breaking away from the default approach is generally not warranted. Indeed, (1) the alternative approach is not more robust, (2) the current default approach is conservative, and (3) there are more pressing challenges for the LCA community to meet. This being said, the study focused on ecoinvent, which should normally be used as a background database. The difference in dispersion between the two approaches may be important in some contexts, and calculating the uncertainty of tree models as a sensitivity analysis could be useful.

     

Characterization of multilayer-enhanced surface-enhanced raman scattering (SERS) substrates and their potential for SERS nanoimaging

Multilayer gold surface-enhanced Raman scattering (SERS) substrates, which consist of continuous gold films that are separated by self-assembled monolayers (SAMs) and cast over 430-nm diameter silica nanospheres on a glass slide, have been evaluated as a means of further enhancing the SERS signals produced from conventional metal film over nanostructure substrates. Evaluation of the effect of various SAMs, with different terminal functional groups, on the SERS enhancement factor were measured and compared to conventional single-layer gold film over nanostructure substrates, revealing relative enhancements as great as 22.4-fold in the case of 2-mercapto-ethanol spacer layers. In addition to evaluation of the effect of different terminal functionalities, the effect of spacer length was also investigated, revealing that the shorter chain length alcohols provided the greatest signals. Employing the optimal SERS multilayer geometry, SERS nanoimaging probes were fabricated and the SERS enhancement factor and variability in enhancement factor were measured over the SERS active imaging area, providing absolute enhancements similar to previous silver-based SERS nanoimaging probes (i.e., 1.2 × 10⁸). Varying the size of the multilayer gold islands that were deposited on the tip of the SERS active nanoimaging probe, it is possible to tune the optimal SERS excitation wavelength accurately and predictably over the range of approximately 450 to 600 nm, without coating the entire surface of the probe and significantly reducing the transmission and resulting signal-to-noise ratio of the images obtained.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

ABSTRACT

Microalgae have enormous potential as feedstock for biofuel production compared with other sources, due to their high areal productivity, relatively low environmental impact, and low impact on food security. However, high production costs are the major limitation for commercialization of algal biofuels. Strategies to maximize biomass and lipid production are crucial for improving the economics of using microalgae for biofuels. Selection of suitable algal strains, preferably from indigenous habitats, and further improvement of those ‘platform strains’ using mutagenesis and genetic engineering approaches are desirable. Conventional approaches to improve biomass and lipid productivity of microalgae mainly involve manipulation of nutritional (e.g. nitrogen and phosphorus) and environmental (e.g. temperature, light and salinity) factors. Approaches such as the addition of phytohormones, genetic and metabolic engineering, and co-cultivation of microalgae with yeasts and bacteria are more recent strategies to enhance biomass and lipid productivity of microalgae. Improvement in culture systems and the use of a hybrid system (i.e. a combination of open ponds and photobioreactors) is another strategy to optimize algal biomass and lipid production. In addition, the use of low-cost substrates such as agri-industrial wastewater for the cultivation of microalgae will be a smart strategy to reduce production costs. Such systems not only generate high algal biomass and lipid productivity, but are also useful for bioremediation of wastewater and bioremoval of waste CO₂. The aim of this review is to highlight the advances in the use of various strategies to enhance production of algal biomass and lipids for biofuel feedstock.     

The positively cooperative binding of concanavalin a to corn starch: The isotherm of binding and a measure of the cooperativity

The binding of concanavalin A to corn starch was investigated by fluorimetric assay. The extent of binding varied linearly with the mass of ligand, and followed a hyperbolic law with respect to the mass of starch. This led to an isotherm of binding: r = 0.33A_oME_o^?0.88, where r is the extent of binding, A_o is the mass of concanavalin A present (both bound and unbound), and M_o is the mass of starch. These results, and Scatchard plots of the data, showed the binding to be positively cooperative. The exponent of the M_o term was shown to be a measure of cooperativity. The binding was dependent on the ionic strength of the dispersion medium, and this indicated that the binding may have an electrostatic component.     

Screening of cultivars for tissue culture response and establishment of genetic transformation in a high-yielding and disease-resistant cultivar of Theobroma cacao

A highly efficient transformation protocol is a prerequisite to developing genetically modified and genome-edited crops. A tissue culture system spanning culture initiation from floral material to conversion of embryos to plants has been tested and improved in Theobroma cacao. Nine cultivars were screened for their tissue culture response and susceptibility to Agrobacterium transfer-DNA delivery as measured through transient expression. These key factors were used to determine the genetic transformability of various cultivars. The high-yielding, disease-resistant cultivar INIAPG-038 was selected for stable transformation and the method was further optimized. Multiple transgenic events were produced using two vectors containing both yellow fluorescent protein and neomycin phosphotransferase II genes. A two-fold strategy to improve both T-DNA delivery and secondary somatic embryogenesis rates was conducted to improve overall transformation frequency. The use of Agrobacterium strain AGL1 and cotyledon tissue derived from secondary somatic embryos ranging in size between 4 to 10 mm resulted in the highest T-DNA delivery efficiency. Furthermore, the use of higher concentrations of basal salts and cupric sulfate in the medium increased the frequency of explants producing greater than ten embryos by five-fold and four-fold during secondary somatic embryogenesis, respectively. Consequently, an optimal combination of all these components resulted in a successful transformation of INIAPG-038 with 3.7% frequency at the T₀ plant-level. Grafting transgenic scions with undeveloped roots to non-transgenic seedlings with healthy roots helped make plantlets survive and facilitated quick transplantation to the soil. The presented strategy can be applied to improve tissue culture response and transformation frequency in other Theobroma cacao cultivars.

     

Receptor-Agonist Interactions in Service-Theoretic Perspective,Effects of Molecular Timing on the Shape of Dose-Response Curves

Service-theoretic concepts and methods, widely used in other fields (e.g., telecommunication and operations research), are useful also in a biochemical setting because the treatment of biocatalysts (enzymes, receptors) as servers and their ligands as customers, based on the established formal methods of service or queuing theory, may lead to insights and results unobtainable by conventional, mass-action-law-based theories. In this article, we apply the service-theoretic approach to receptor-agonist systems and show how by changing the stochastic time pattern of “operationally relevant” point events (e.g., instants of agonist arrival, instants of postclimax agonist departure) a great variety of dose-response curves may be generated, even in very simple reaction schemes, which, according to mass action kinetics, invariably lead to hyperbolic r(A) curves (r and A stand for response and agonist concentration, respectively). The molecular timing inherent to a hyperbolic response system is not optimal: for instance, at the agonist concentration A₅₀, half of the agonist molecules are rejected (“lost”) because of unfortunate timing of the arrival events. The fraction of lost arrivers can be diminished considerably if the arrivals are better timed: “sub-Poisson” arrivals improve the timing and, thus, convert hyperbolic r(A) curves into “lifted” nonhyperbolic ones. Conversely, “super-Poisson” arrivals make the nonoptimal timing in hyperbolic response systems even worse and, thus, convert hyperbolic r(A) curves into “depressed” nonhyperbolic ones. Furthermore, under special timing conditions, nonhyperbolic r(A) curves can be generated, which are partly lifted, partly depressed relative to the reference hyperbola, and which resemble in shape well-known nonhyperbolic forms of enzyme and receptor kinetics (negatively cooperative, positively cooperative, and sigmoidal kinetics). In addition unusual (undulatory and sawtooth-like) r(A) curves can be generated solely by changing the temporal pattern of arrival and service completion instants. Virtually any shape of dose-response curves may be obtained by allowing for probability distributions whose characteristic shape varies with their mean; we call such distributions “variomorphic” and apply them to the arrival process of agonist molecules.     

Errors in the determination of low levels of nitrate in lake sediments

Abstract

Error sources associated with the spectrophotometric determination of low levels (e.g. <2 μg g^?1) of nitrate in sediments have been examined and problems identified included incomplete nitrate recovery (attributable in part to anion resorption) and light scattering by colloidal (<0.45 μm) matter in extract solutions (minimised by using uncoloured extract in the reference beam). Optimum retrieval (>90%) of nitrate from the marine lake sediments studied was achieved with 15 min mixing with 0.1 M NH₄CI, using a sediment to extractant ratio of 1:30. The nitrate in the extracts was determined by reducing it to nitrite (using Cd powder), with subsequent colour development based on the addition of sulfanilic acid and N-1-naphthyl-ethylenediamine dihydrochloride. The reduction step was sensitive to the experimental conditions used, but was near quantitative using 0.1 M NH₄CI extracts. (Much lower transformation levels were observed when the nitrate solutions contained KCI or CaSO₄, or when Zn powder was used as the reductant). All the sediments tested sorbed nitrate ion from solution (some very avidly) and this sorbed ion was not readily retrieved by back extraction into NH₄CI solutions.     

Interfacial Charge Modulation: An Efficient Strategy for Boosting Spatial Charge Separation on Semiconductor Photocatalysts

Surface modulation via injection or extraction of charge carriers in microelectric devices has been used to tune the energy band alignment for desired electrical and optical properties, yet not well recognized in photocatalysis field. Here, taking semiconductor bismuth tantalum oxyhalides (Bi₄TaO₈X) as examples, chemically inactive molybdenum oxide (MoO₃) with a large work function is introduced to qualitatively tune the properties of interfacial charges, achieving an evidently enhanced upward band bending and intensive built‐in electric field. Such a simple charge modulation exhibits a remarkable improvement in photocatalytic water oxidation, reaching an apparent quantum efficiency of 25% at the input wavelength of 420 nm. The validity and generality of surface charge modulating strategy are further demonstrated using other semiconductors (e.g., C₃N₄) and decorators (e.g., V₂O₅). The findings not only provide a promising strategy for rationally manipulating the interfacial built‐in electric field in photocatalysis but also pave the way to learn from microelectronic technologies to construct artificial photosynthesis systems for solar energy conversion.     

Microbial Community Structure and Metabolic Potential of the Hyporheic Zone of a Large Mid-Stream Channel Bar

Abstract

To develop a greater understanding of hyporheic zone microbial biogeochemistry, we sampled pore fluids from a piezometer array associated with the McCarran Ranch channel bar (MRCB); a partially submerged cobble island in the Truckee River, NV, USA. Flowing surface water and pumped pore fluids were characterized by prokaryotic community structure, metabolic potential, and aqueous physicochemistry. Concentrations of potential respiratory electron acceptors were highest in surface water and riverbed porewater and sequentially depleted in porewaters along the inferred flowpath (O₂, then NO₃^?, then SO₄^2?). Correspondingly, cultivable nitrate reducers/denitrifiers were most abundant in surface water and riverbed porewater, despite oxic conditions. Cultivable sulfate reducers were overall most abundant in surface water. Prokaryotic community reconstruction from 16S rRNA gene sequences indicates that the surface water community was less diverse than that of porewater and supports a shift in metabolic strategy, from aerobic heterotrophy in surface water (e.g., Comamonadaceae and Sporichthyaceae) to chemolithotrophy and anaerobic metabolisms (e.g., Hydrogenophaga spp., Ferribacterium spp., Methanobacterium spp.) along the hyporheic flow path. These data indicate that prokaryotic communities within the MRCB are phylogenetically and metabolically diverse and contribute to biogeochemical cycling in this common yet relatively understudied habitat.     

Managing microbial communities in membrane biofilm reactors

Membrane biofilm reactors (MBfRs) deliver gaseous substrates to biofilms that develop on the outside of gas-transfer membranes. When an MBfR delivers electron donors hydrogen (H₂) or methane (CH₄), a wide range of oxidized contaminants can be reduced as electron acceptors, e.g., nitrate, perchlorate, selenate, and trichloroethene. When O₂ is delivered as an electron acceptor, reduced contaminants can be oxidized, e.g., benzene, toluene, and surfactants. The MBfR’s biofilm often harbors a complex microbial community; failure to control the growth of undesirable microorganisms can result in poor performance. Fortunately, the community’s structure and function can be managed using a set of design and operation features as follows: gas pressure, membrane type, and surface loadings. Proper selection of these features ensures that the best microbial community is selected and sustained. Successful design and operation of an MBfR depends on a holistic understanding of the microbial community’s structure and function. This involves integrating performance data with omics results, such as with stoichiometric and kinetic modeling.

     

Partial nitritation at elevated loading rates: design curves and biofilm characteristics

There is a need to develop low operational intensity, cost-effective, and small-footprint systems to treat wastewater. Partial nitritation has been studied using a variety of control strategies, however, a gap in passive operation is evident. This research investigates the use of elevated loading rates as a strategy for achieving low operational intensity partial nitritation in a moving bed biofilm reactor (MBBR) system. The effects of loading rates on nitrification kinetics and biofilm characteristics were determined at elevated, steady dissolved oxygen concentrations between 5.5 and 7.0 mg O₂/L and ambient temperatures between 19 and 21 °C. Four elevated loading rates (3, 4, 5 and 6.5 g NH₄⁺-N/m² days) were tested with a distinct shift in kinetics being observed towards nitritation at elevated loadings. Complete partial nitritation (100% nitrite production) was achieved at 6.5 g NH₄⁺-N/m² days, likely due to thick biofilm (572 µm) and elevated NH₄⁺-N load, which resulted in suppression of nitrite oxidation.

     

Broadband Optical Reflection Modulator in Indium-Tin-Oxide-Filled Hybrid Plasmonic Waveguide with High Modulation Depth

A surface plasmon resonance (SPR)-based optical reflection modulator consisting of vertically stacked silica-silicon-HfO₂-ITO-HfO₂-Ag-prism multilayer is proposed and numerically investigated. The free carrier-concentration-dependent permittivity of indium-tin-oxide (ITO) at the HfO₂/ITO interface induces an epsilon-near-zero (ENZ) effect contributing to strong field enhancement and modifies the SPR condition of incident light. With optimal geometry parameters and proper design of carrier concentration at the accumulation layer, modulation depth (MD) of ~100% and insertion loss (IL) of 3.7% can be simultaneously achieved. The IL can be further reduced by engineering silicon layer thickness. Moreover, the device offers a broadband operation wavelength from 1.5 to 1.6 μm with the variations of MD and IL smaller than 4 and 3%, respectively.

     

Detecting Photoactivation of Phosphoenolpyruvate Carboxylase in C(4) Plants : An Effect of pH

Photoactivation of phosphoenolpyruvate carboxylase in C₄ plants is detected more efficiently when activity is assayed at suboptimum pH (e.g. 7.2); the magnitude of the light effect is often larger at low phosphoenolpyruvate concentration.

Darkness and low assay pH induce an allosteric behavior (positive cooperativity with phosphoenolpyruvate) which is relieved in light or by higher pH; thus, normal Michaelis-Menten kinetics are exhibited only when the enzyme is extracted during the day and assayed at pH 8.2.

Light activation, pH, and substrate level appear to be components of a regulatory device suppressing the activity in darkness and enhancing it under light.

     

Reversible H-Atom Abstraction from Alcohols by Thiyl Radicals: Determination of Absolute Rate Constants by Pulse Radiolysis

Hahn-Meitner-Institut Berlin, Bereich Strahlenchemie, Glienicker Str. 100, 1000 Berlin 39, Fed. Rep. Germany

Penicillamine thiyl radicals, PenS', are shown to abstract hydrogen atoms from 2-propanol and to establish an equilibrium

PenS' + (CH₁)₂ CHOH ? PenSH + (CH₃)₂ COH.

The rate constants for the forward and back reaction have been determined to (1.4 ± 0.3) × 10⁴ and (1.2 ± 0.3) × 10⁸ M^-1 s^-1, respectively, by means of pulse radiolysis. The data have been obtained from various independent methods which include direct measurements and competitive schemes involving irreversible interception of the alcohol radical by electron acceptors (e.g. CCl₄. PNAP) and/or the thiyl radical by antioxidants (e.g. α-tocopherol). The results demonstrate that the reaction of carbon-centered radicals with thiols, in radiation biology commonly known as “repair” reaction, may be reversed and thus imply the possibility of thiyl radical induced biological damage.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine by using microgel beads containing Pseudomonas cepacia lipase

Abstract

Pseudomonas cepacia lipase (PCL) was immobilized in alginate microgel beads by electrostatic dispersion. The high electrical potential applied in the immobilization process could significantly decrease the droplet size. The optimum conditions for lipase immobilization were 2% (w/v) alginate, 100 mM CaCl₂, 8 mg/mL enzyme, 4 kV electrical potential and 200 μm mean bead size. Under these conditions, 78.2 U/g of immobilized PCL activity was obtained with 39.1% retained activity and 57.2% immobilization efficiency. The immobilized PCL (PCL-CA) was subsequently used in the enantioselective hydrolysis of (R, S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester. Although PCL-CA exhibited slightly lower activity than free PCL, it preserved the high enantioselectivity (E-value >?200), which afforded enantiomerically pure (R)-acid (99% e.e._p). Furthermore, PCL-CA exhibited higher thermal stability, storage and medium stability than that of free PCL. Batch-wise operational stability studies demonstrated that PCL-CA retained its initial activity for at least 10 cycles of hydrolysis.