RpoH(II) activates oxidative-stress defense systems and is controlled by RpoE in the singlet oxygen-dependent response in Rhodobacter sphaeroides

Photosynthetic organisms need defense systems against photooxidative stress caused by the generation of highly reactive singlet oxygen (¹O₂). Here we show that the alternative sigma factor RpoH_II is required for the expression of important defense factors and that deletion of rpoH_II leads to increased sensitivity against exposure to ¹O₂ and methylglyoxal in Rhodobacter sphaeroides. The gene encoding RpoH_II is controlled by RpoE, and thereby a sigma factor cascade is constituted. We provide the first in vivo study that identifies genes controlled by an RpoH_II-type sigma factor, which is widely distributed in the Alphaproteobacteria. RpoH_II-dependent genes encode oxidative-stress defense systems, including proteins for the degradation of methylglyoxal, detoxification of peroxides, ¹O₂ scavenging, and redox and iron homeostasis. Our experiments indicate that glutathione (GSH)-dependent mechanisms are involved in the defense against photooxidative stress in photosynthetic bacteria. Therefore, we conclude that systems pivotal for the organism's defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by RpoH_II in R. sphaeroides.     

Electrospray identification of new polyoxochromate species

Electrospray ionization mass spectrometry (ESI MS) has been conducted on the ammonium and alkali metal (A=Li⁺, Na⁺ and K⁺) dichromate systems. A large number of previously unknown polyoxochromate species have been characterized. Major series that have been identified include [A_x+1H_xCr^VI_xO_4x]⁺ (Li⁺, x=1-5; Na⁺, x=1-7; K⁺, x=1-4) and [A_2x−1Cr^VI_xO_4x−1]⁺ (Li⁺, x=2, 3; Na⁺, x=2-4; K⁺, x=2, 3) in the alkali metal dichromate systems, and [HCr^VI_xO_3x+1]⁻ (x=1-5) in the ammonium dichromate system. Several series also contain mixed oxidation state species, ranging from Cr(V) to Cr(II) in conjunction with Cr(VI), which is consistent with the ease of reduction of Cr(VI). Negative ion ESI MS spectra clearly demonstrate the existence of [HCrO₄]⁻ as the most abundant ion at −20 V, suggesting that its existence in solution is not just hypothetical, as was previously thought. The polymerization units for the series observed include {AHCrO₄}, {A₂CrO₄} and {CrO₃}, with the latter prominent in the alkali metal systems. This presumably arises from the fragmentation of dichromate, A₂Cr₂O₇→{A₂CrO₄}+{CrO₃}. Moreover, the ESI MS of the dichromate compounds have illustrated that the preservation of tetrahedral stereochemistry is of paramount importance for these systems, which leads to only limited polymerization compared to the related molybdate and tungstate systems.     

Activation of a Na+-dependent amino acid transport system in preimplantation mouse embryos

The uptake of l-methionine-methyl-³H and l-leucine-³H from completely defined medium into acid-soluble fractions of preimplantation mouse embryos has been studied. Late four-cell embryos and early blastocysts raised in vitro can concentrate both amino acids by processes which exhibit saturable, Michaelis-Menten type kinetics, characteristic of carrier-mediated active transport systems. This uptake is temperature-sensitive and inhibited by certain amino acids which compete for the same uptake sites. Methionine uptake seems to be mediated by a single transport system (K_m = 6.25 × 10^?5M) at the four-cell stage. Complex kinetics suggest that two distinct transport systems exist at the early blastocyst stage (K_m = 6.25 × 10^?5M; 8.9 × 10^?4M). V_max values (mg/embryo/15 min) for methionine and leucine transport increase significantly from the late four-cell stage to the blastocyst stage, suggesting that additional carriers are produced or activated during development.Most importantly, leucine and methionine transport is Na⁺-independent at the four-cell stage, methionine transport is partially dependent at the morula stage, and both amino acids are completely Na⁺-dependent at the blastocyst stage. The cumulative results suggest that preimplantation embryos accumulate leucine and methionine by specific, chemically mediated, active transport systems. The qualitative and quantitative developmental changes in cell membrane function may represent preparatory steps for subsequent growth of embryonic and/or trophoblastic cells.     

A yeast arginine specific tRNA is a remnant aspartate acceptor

High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNA^Asp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA₄^Arg. This tRNA has a sequence strikingly similar to that of tRNA^Asp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA₄^Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA₄^Arg into an aspartate acceptor, as efficient as tRNA^Asp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA₄^Arg. The latent aspartate acceptor capacity in a contemporary tRNA^Arg leads to the proposal of an evolutionary link between tRNA₄^Arg and tRNA^Asp genes.     

Effects of increasing nutrient supply and omnivorous feeding on the size spectrum slope: a size-based nutrient-phytoplankton-zooplankton model

Size spectrum slope has been considered as an indicator for system productivity and size-dependent trophic interactions in plankton communities. We investigated the effect of new nutrient flux (N _in ) and feeding breadth of zooplankton (σ²) on spectral slopes of the plankton community using a size-structured nutrient-phytoplankton-zooplankton model. We studied two types of plankton systems (1) at a steady state and (2) with fluctuating plankton populations. For the steady state systems, the spectral slopes increased with N _in and σ². In contrast, for the systems in fluctuation, spectral slopes constructed from long-term average plankton populations decreased with increasing N _in and σ². In addition to spectral slopes constructed from long-term average population densities, we investigated slope variability in systems with fluctuating plankton populations. The variability of spectral slopes was found to increase with N _in and σ², indicating that spectral slope variation is large in a eutrophic system or when zooplankton feed on a wide size range of prey. Empirically, because N _in is difficult to measure, nutrient concentration is investigated instead. Our model predicts that the spectral slope is positively correlated with nutrient concentration due to the accumulated mesozooplankton biomass, which was confirmed by our study in plankton community in a subtropical reservoir in Taiwan.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

Kinetics of oxidation of ferrocene by tris-1,10-phenanthrolinecobalt(III) in t-butyl alcoholwater and acetonewater mixtures

Rate constants and activation parameters (ΔH^≠ and ΔS^≠)are reported for the oxidation of ferrocene by the tris-1,10-phenanthrolinecobalt(III) cation in t-butyl alcoholwater and in acetonewater solvent mixtures. Solvent effects on reactivity trends for these systems, for this same reaction in methanolwater mixtures, and for cobalt(II)-catalysed racemisation of Co(phen)_3³⁺ in t-butyl alcoholwater solvent mixtures are analysed into initial state and transition state contributions. The dependences of solubilities on solvent composition for ferrocene and for [Co(phen)₃](ClO₄)₃ in methanol, t-butyl alcohol, and acetonewater mixtures are also reported; these results are needed in order to establish solvent effects on the initial states of the reactions studied.     

Diploidy and repair of radiation damage in Saccharomyces mutants

Diploid wild-type cells of Saccharomyces and cells homozygous for one of the single genes r^s₁, r^s₂ of the two alleles of the r^s₃ gene (rad 2 gene) were tested for their response to ionizing irradiation in the presence and absence of oxygen. Both immediate and delayed plating (agar-holding) techniques were used.An identical oxygen enhancement ratio (OER) of 2.5 was observed in r^s₁r^s₁, r^s₂r^s₂ cells and the wild type. In r^s_3?1r^s_3?1 and r^s_3?2r^s_3?2 cells the OER was reduced. Their sensitivity to ionizing irradiation in oxic conditions was the same as that of the wild type.r^s₂r^s₂ cells showed an increase in relative sensitivity by a factor of 4 in both conditions of irradiation. The relative sensitivity of r^s₁r^s₁ cells was increased by a factor of 2.5 in hypoxic conditions and the sensitivity in hypoxia was identical to that of wild type cells in oxic conditions. In contrast to haploid r^s₁ cells the oxygen effect was fully expressed in diploid r^s₁r^s₁ cells.The results on diploid strains are discussed in terms of three independent genetically determined repair systems in Saccharomyces. The expression of these systems is influenced by ploidy.     

Nuclear Magnetic Resonance of Tissue 23Na: II. Theoretical Line Shape

The theoretical line-shape function of the nuclear magnetic resonance (NMR) signal of ²³Na in biological tissue (and other unoriented systems) was obtained under the following conditions: (I) there occur two states of ²³Na in the system, (II) the exchange of ²³Na between the two states is rapid (but not too rapid), (III) in the absence of exchange, the ²³Na in one state is characterized by a single transverse relaxation time T₂ and a single Larmor frequency, and (IV) in the absence of exchange, the ²³Na in the other state possesses (a) two different values of T₂ and/or (b) more than one Larmor frequencies in the first order perturbation effect. The theoretical signal obtained consists of two Lorentzian components, which are centered at the same frequency, but characterized by different T₂. Only the narrower component, comprising 40% of the total intensity, is visible, when the fast T₂ is sufficiently short. The theoretical line-shape function of ²³Na signal was also calculated for oriented systems in which the above conditions are fulfilled.     

1H NMRD studies of solutions of paramagnetic metal ions in ethyleneglycol

Solvent¹H T^?1₁ values at magnetic fields between 2.3 × 10^?4 and 1.2 T were measured for ethyleneglycol solutions of manganese(II), cobalt(II), nickel(II), copper(II), and gadolinium(III) salts in the temperature range ?10–+40 °C. The T^?1₁ profiles were interpreted on the ground of the available theories according to whether the electronic relaxation times or the rotational times are the correlation times for the interaction. The comparison among systems experiencing different correlation times and electronic structures (metal ions in water, ethyleneglycol, and in proteins) allowed us to test the general theoretical approach regarding nuclear relaxation times in paramagnetic systems. Such theoretical approaches allow researchers to extract structural parameters and information on the correlation times and dynamic processes operative in the various investigated systems.     

Hemoglobin-water interactions in normal and sickle erythrocytes by proton magnetic resonance T1ϱ measurements

The dependence of the water proton magnetic resonance spin-lattice relaxation rate (T_1?^?1) in the rotating frame on the strength of the spin-locking (H₁) field has been investigated for packed oxy and deoxy normal and sickle erythrocytes at temperatures from 9 to 40 °C. The T_1?^?1 of oxy or deoxy normal erythrocytes shows no dependence on H₁ up to ~7 G at any temperature studied. On the other hand, T_1?^?1 decreases from about 40 s^?1 to 15 s^?1 (H₁ from 0 to ~7 G) for deoxygenated packed sickle cells at 40 °C. The magnitude of this variation of T_1?^?1 with H₁ decreases with decreasing temperature. Oxy packed sickle cells also show a dependence of T_1?^?1 on H₁ but the magnitude is <10% of that of the deoxygenated samples. These results suggest that water proton T_1?^?1 measurements are a sensitive probe of hemoglobin S polymerization and provide a novel technique for the study of slow water motions in these systems. The T_1?^?1 results are compared with low frequency T₁^?1 results of other investigators on hemoglobin S solutions. Analysis of the data suggests that water proton motions with correlation times of the order of 10^?5 s are present in the deoxygenated sickle cell samples at temperatures above 10 °C.     

Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P_i). To better understand phosphorus movement between the bacteroid and the host plant, P_i transport was characterized in R. tropici. We observed two P_i transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K_m and V_max values for the low-affinity system were estimated to be 34 ± 3 μM P_i and 118 ± 8 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively, and the K_m and V_max values for the high-affinity system were 0.45 ± 0.01 μM P_i and 86 ± 5 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively. Both systems were inducible by P_i starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P_i transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the P_i transport rate was correlated with the intracellular ATP concentration. Also, P_i movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P_i transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.     

Feasibility of using water-immiscible organic solvents in biological waste-gas treatment

In order to conclude about the feasibility of using water-immiscible organic solvents in biological waste-gas treatment, a theoretical study was done in which different types of organic-solvent-containing systems are compared with systems where the pollutant is transferred directly to the water phase. For each system the total equipment volume needed to remove 99% of a pollutant from a waste-gas stream is calculated. Three different pollutants with a different solubility in water are considered: Hexane (m _gw =71), dichloromethane (m _gw =0.1) and acetone (m _gw =0.0016), withm _gw the partition coefficient (kg/m³ gas/kg/m³ water) of the pollutant between the gas and the water phase. From the results it is concluded that the use of organic solvents is only advantageous in case the specific area for mass transfer between solvent and water is large enough to compensate for the additional transport resistance introduced by the solvent, and secondly if the solvent shows a sufficiently high affinity for the pollutants.     

N2O Evolution by Green Algae

Evidence is presented here that axenic cultures of Chlorella, Scenedesmus, Coelastrum, and Chlorococcum spp. evolve N₂O when grown on NO₂⁻, showing that the Chlorophyceae are a source of N₂O in aquatic systems.     

Is it always correct to use the Marcus cross relation for calculations of electron self-exchange rates?

The rate constant of the electron self-exchange reaction, which proceeds via the outer sphere mechanism, k₁₁, of a redox couple, reflects the basic tendency of the reaction to participate in redox processes. Often k₁₁ is derived from the rate constant of a redox reaction, k₁₂, by applying the Marcus cross relation: k₁₂ = (k₁₁k₂₂K₁₂f₁₂)^1/2W₁₂. This derivation is based on the assumption that the products of the cross reaction are formed in their ground states. However, the k₁₁ values obtained by this method for several redox systems, e.g. for [Co(NH₃)₆]^3+/2+, Cu^2+/+(aq) and Eu^3+/2+(aq), depend strongly on the redox reaction studied for its derivation. It is proposed that these discrepancies are due to the formation, in some of these reactions, of products in vibrationally excited states and/or as isomers of the final products. Thus, the lowest value of k₁₁ obtained experimentally is either correct or an upper limit for the correct value if correct k₂₂ are used.     

Stable Isotope Probing with 15N2 Reveals Novel Noncultivated Diazotrophs in Soil

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of nifH gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel ¹⁵N₂-DNA stable isotope probing (⁵N₂-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from ¹⁵N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the Rhizobiales while the other two represent deeply divergent lineages of noncultivated bacteria within the Betaproteobacteria and Actinobacteria, respectively. Analysis of nifH genes from ¹⁵N-labeled DNA also revealed three microbial groups, one of which was associated with Alphaproteobacteria while the others were associated with two noncultivated groups that are deeply divergent within nifH cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that ¹⁵N₂-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by Actinobacteria outside of the order Actinomycetales.     

Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3

Dynamic structures of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers induced in oriented lipid membranes, which are interacting with membrane-acting antimicrobial peptides (AMPs), magainin-2 and aurein-3.3, were explored by ³¹P and ²H solid-state NMR (ssNMR) spectroscopy. Various types of phospholipid systems, such as POPC-d₃₁, POPC-d₃₁/POPG, and POPC-d₃₁/cholesterol, were investigated to understand the membrane disruption mechanisms of magainin-2 and aurein-3.3 peptides at various peptide-to-lipid (P:L) ratios. The experimental lineshapes of anisotropic ³¹P and ²H ssNMR spectra measured on these peptide-lipid systems were simulated reasonably well by assuming the presence of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers, in membranes. Furthermore, the observed decrease in the anisotropic frequency span of either ³¹P or ²H ssNMR spectra of oriented lipid bilayers, particularly when anionic POPG lipids are interacting with AMPs at high P:L ratios, can directly be explained by a thinned membrane surface model with fast lateral diffusive motions of lipids. The spectral analysis protocol we developed enables extraction of the lateral diffusion coefficients of lipids distributed on the curved surfaces of pores and thinned bilayers on a few nanometers scale.     

Assessment of nitrate leaching loss on a yield-scaled basis from maize and wheat cropping systems

Background and aims

It is so far a gap in knowledge to assess nitrate (NO₃ ^?) leaching loss linking with crop yield for a given cereal cropping system.

Methods

We conducted a meta-analysis on 32 published studies reporting both NO₃ ^? leaching losses and crop yields in the maize (N?=?20) and wheat (N?=?12) systems.

Results

On average, 22 % and 15 % of applied fertilizer N to wheat and maize systems worldwide are leached in the form of NO₃ ^?, respectively. The average area-scaled NO₃ ^- leaching loss for maize (57.4 kg N ha^?1) was approx. two times higher than for wheat (29.0 kg N ha^?1). While, if scaled to crop yields, the average yield-scaled NO₃ ^? losses were comparable between maize (5.40 kg N Mg^?1) and wheat (5.41 kg N Mg^?1) systems. Across all sites, the lowest yield-scaled NO₃ ^? leaching losses were observed at slightly suboptimal fertilization rates, corresponding to 90 % and 96 % of maximum maize or wheat yields, respectively.

Conclusions

Our findings suggest that small adjustments of agricultural N management practices can effectively reduce yield-scaled NO₃ ^? leaching losses. However, further targeted field experiments are still needed to identify at regional scale best agricultural management practices for reducing yield-scaled NO₃ ^? leaching losses in maize and wheat systems.     

Partitioning of amylase produced by Aspergillus niger in solid state fermentation using aqueous two-phase systems

Equilibrium data of aqueous two-phase systems composed of polyethylene glycol (4000 g mol⁻¹ or 6000 g mol⁻¹) and Li₂SO_4, (NH₄)₂SO₄ or Na₂SO₄ at pH 6.5 and 25 °C were obtained. The efficiency of these in the partition of amylases derived from Aspergillus niger was determined. The experimental data of binodal curves and tie lines were used to estimate the group interaction parameters using the UNIFAC model. Additionally, the influence of phases on the activity of the enzymes was investigated. The results indicate that the polymer molar mass did not influence the biphasic region size. However, the cations under study presented differences in induction to phase formation. It was verified that the systems formed with the Na⁺ presented a larger biphasic region. The increase in the molar mass of the polymer caused the increase in the exclusion volume from 3970.732 g mol⁻¹ to 5700.873 g mol⁻¹. The transfer Gibbs free energy of enzymes presented values between −1296.30 kJ mol⁻¹ and −2867.70 kJ mol⁻¹, that is, the process was spontaneous for all systems studied. The systems formed by (NH₄)₂SO₄ and PEG 4000 g mol⁻¹ presented the best K_e result (3.421) and theoretical recovery of 80.35 %.     

Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells

Two active calcium (Ca²⁺) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca²⁺ transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl⁻-stimulated, NO₃⁻-inhibited ATPase activity on sucrose density gradients. Ca²⁺ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N′-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS). The K_m for MgATP and Ca²⁺ were 0.1 mm and 21 micromolar, respectively. The predominant Ca²⁺ transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca²⁺ transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I₅₀ = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca²⁺ dependent kinetics were complex with an apparent K_m ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca²⁺/H⁺ antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca²⁺ transport systems are proposed to restore and maintain cytoplasmic Ca²⁺ homeostasis under changing cellular and environmental conditions.