Visualization of NO3?/NO2? Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System

Nitrate (NO₃⁻) and nitrite (NO₂⁻) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO₃⁻/NO₂⁻ exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO₃⁻/NO₂⁻ for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO₃⁻/NO₂⁻ in physiology), by employing NO₃⁻/NO₂⁻-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO₃⁻ and NO₂⁻, and its FRET signal is changed in response to NO₃⁻/NO₂⁻ in the micromolar range. Furthermore, both an increase and decrease in cellular NO₃⁻ concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO₃⁻/NO₂⁻ dynamics in various organisms, including plants and animals.     

Region-specific rates of molecular evolution: A fourfold reduction in the rate of accumulation of “Silent” mutations in transcribed versus nontranscribed regions of homologous DNA fragments derived from two closely related mouse species

Summary We have sequenced homologous DNA fragments of 2.7 and 2.8 kbp derived from the closely related mouse speciesMus musculus domesticus (M. domesticus) andMus musculus musculus (M. musculus), respectively. These two species diverged approximately 1 million years ago. Each DNA fragment contains 1.35 kbp of the 3′ end of the constitutively expressed 2.2-kbpaprt (adenine phosphoribosyltransferase) gene and a similarly sized nontranscribed region downstream of theaprt gene. Theaprt gene region contains protein coding sequences (0.35 kbp), intronic sequences (0.75 kbp), and a 3′ nontranslated sequence (0.25 kbp). Both theM. domesticus andM. musculus downstream regions share three partial copies of the B1 repetitive element with theM. musculus downstream region containing an additional complete copy of this element. A comparison of the 2.7-and 2.8-kbp DNA fragments revealed a total of 63 molecular alterations (i.e., mutations) that were approximately fourfold more abundant in the nontranscribed downstream region than in the transcribedaprt gene. Of the 11 mutations observed in the transcribed region, 7 were found in introns, 3 in the 3′ untranslated sequence, and 1 was a synonymous change in an exon. A comparison of the human andM. domesticus aprt genes has previously revealed no homology in either the intronic or 3′ nontranslated regions with the exception of a 26-bp sequence in intron 3 and sequences at the exon/intron boundaries necessary for correct mRNA splicing (Broderick et al.,Proc. Natl. Acad. Sci. USA, 84:3349, 1987). Therefore, there does not appear to be selective pressure for sequences within these regions. We conclude that there is a lower rate of accumulation of “silent” mutations in the transcribed mouseaprt gene than in a contiguous nontranscribed downstream region. A possible molecular mechanism involving preferential DNA repair for the transcribed region is discussed.     

Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using 13NO3?

¹³N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs.     

The solid state chemistry of uranium. Part I: The thermal decomposition of U(NO3)4·2tdpo and U(NO3)4·2tppo

The thermal decomposition of U(NO₃)₄·2tdpo (tdpo=tris-(dimethylamino)phosphine oxide) and U(NO₃)₄·2tppo (tppo=triphenyl phosphine oxide have been examined using thermogravimetry and differential scanning calorimetry. Decomposition of the crystals approximates to the general reaction U(NO₃)₄·2L(s) → UO₂(NO₃)₂·2L(s) + gases The general thermal behaviour of the two compounds were found to be different.     

Cloning of AAC(3) and AAD(2″) genes fromAcinetobacter: differential expression in the host strain

The mechanism of resistance to gentamicin and tobramycin in a clinical isolate ofAcinetobacter baumannii, in which aminoglycoside-modifying enzymes were not detected, was investigated. For increase of the resistance gene product, DNA prepared from theA. baumannii isolate was cloned into pUC18 and introduced intoEscherichia coli by transformation. Gentamicin-resistant transformants were screened for aminoglycoside-modifying enzymes. This approach identified two genes encoding AAC(3) and AAD(2) activity, respectively. To determine whether both genes are expressed in the hostAcinetobacter strain, we extracted total cellular RNA from this strain, and Northern blots were hybridized with the cloned AAC(3) and AAD(2) structural genes. mRNA transcribed from the AAC(3) gene alone was detected. This shows that cloning a functional resistance gene is not sufficient in itself to investigate mechanisms of resistance in bacterial strains without detectable aminoglycoside-modifying activity. Furthermore, this study suggests a potential limitation of antibiotic resistance gene probes for studying mechanisms of resistance.     

Nitrogen Uptake by Wheat Seedlings,Interactive Effects of Four Nitrogen Sources: NO3?, NO2?, NH4+, and Urea

The net influx (uptake) rates of NO₃⁻, NH₄⁺, NO₂⁻, and urea into roots of wheat (Triticum aestivum cv Yecora Rojo) seedlings from complete nutrient solutions containing all four compounds were monitored simultaneously. Although urea uptake was too slow to monitor, its presence had major inhibitory effects on the uptake of each of the other compounds. Rates of NO₃⁻, NH₄⁺, and NO₂⁻ uptake depended in a complex fashion on the concentration of all four N compounds. Equations were developed which describe the uptake rates of each of the compounds, and of total N, as functions of concentrations of all N sources. Contour plots of the results show the interactions over the range of concentrations employed. The coefficients of these equations provide quantitative values for evaluating primary and interactive effects of each compound on N uptake.     

Genetic deletion of laminin isoforms β2 and γ3 induces a reduction in Kir4.1 and aquaporin-4 expression and function in the retina

Background

Glial cells such as retinal Müller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir) channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Müller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Müller cells.

Methodology/Principal Findings

The aim of the present study was to test the function of the β2 and γ3 containing laminins in murine Müller cells. We used knockout mice with genetic deletion of both β2 and γ3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a down-regulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Müller cells from laminin β2 and γ3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice.

Conclusion

These findings demonstrate a strong impact of laminin β2 and γ3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Müller cells.     

High supply of NO3 ? mitigates salinity effects through an enhancement in the efficiency of photosystem II and CO2 assimilation in Jatropha curcas plants

This study was performed to determine if a high supply of N-NO₃ ^? is capable of mitigating negative salinity effects on photosynthesis and growth through the stimulation of nitrate assimilation, which could act as an sink from photosynthetic electron transport chain and restrict the over reduction in thylakoid membrane in Jatropha curcas leaves. The experiment was arranged in a factorial design with two nitrate concentrations (1 and 10?mM) and two NaCl levels (0 and 100?mM). Salt-stressed plants supplied with high NO₃ ^? demonstrated a higher nitrate uptake rate, nitrate reductase activity and soluble-protein content when compared with plants that presented low nitrate uptake. High nitrate assimilation was associated with higher leaf growth, CO₂ assimilation and lower membrane damage in salt-stressed plants. The superior performance of salt-stressed plants grown with high NO₃ ^? was indicated by a higher effective quantum yield of PSII and electron transport rate and lower energy excess at the PSII level and non-photochemical quenching. Interestingly, a high NO₃ ^? level in the absence of NaCl did not alter the leaf growth, photochemical activity and gas exchange parameters when compared with plants supplied with low nitrate. The proline and glycinebetaine contents were similarly increased in both low- and high-NO₃ ^? salt-stressed plants. Our data suggest that the favorable effects induced by high nitrate supply were possibly associated with stimulation in the nitrate assimilatory pathway. This process might have acted as a sink of electrons from the thylakoid membranes minimizing photo-damage and stimulating CO₂ assimilation under salinity in J. curcas.     

Binding of ReO4 ? with an engineered MoO4 2?-binding protein: towards a new approach in radiopharmaceutical applications

Radiolabeled biomolecules are routinely used for clinical diagnostics. ^99mTc is the most commonly used radioactive tracer in radiopharmaceuticals. ¹⁸⁸Re and ¹⁸⁶Re are also commonly used as radioactive tracers in medicine. However, currently available methods for radiolabeling are lengthy and involve several steps in bioconjugation processes. In this work we present a strategy to engineer proteins that may selectively recognize the perrhenate (ReO₄ ⁻) ion as a new way to label proteins. We found that a molybdate (MoO₄ ²⁻)-binding protein (ModA) from Escherichia coli can bind perrhenate with high affinity. Using fluorescence and isothermal titration calorimetry measurements, we determined the dissociation constant of ModA for ReO₄ ⁻ to be 541 nM and we solved a crystal structure of ModA with a bound ReO₄ ⁻. On the basis of the structure we created a mutant protein containing a disulfide linkage, which exhibited increased affinity for perrhenate (K _d = 104 nM). High-resolution crystal structures of ModA (1.7 ?) and A11C/R153C mutant (2.0 ?) were solved with bound perrhenate. Both structures show that a perrhenate ion occupies the molybdate binding site using the same amino acid residues that are involved in molybdate binding. The overall structure of the perrhenate-bound ModA is unchanged compared with that of the molybdate-bound form. In the mutant protein, the bound perrhenate is further stabilized by the engineered disulfide bond.     

Effects of Al on nitrogen (NH 4 + and NO 3 − ) uptake,nitrate reductase activity and proton release in two sorghum cultivars differing in Al tolerance

After growth for 17 to 36 days on nutrient solutions with NH₄NO₃ as nitrogen source (pH 4.2) dry matter of sorghum genotype SC0283 was much less affected by Al (1.5 and 3.0 ppm) than that of genotype NB9040. In the absence of Al both cultivars released protons into the nutrient solution as a result of an excess of cationic nutrients taken up. When Al was present, this proton efflux per unit dry weight increased drastically, especially with the sensitive genotype NB9040. Chemical analysis of plant material and continuous analyses of NO ₃ ⁻ and NH ₄ ⁺ in the nutrient solution indicated, that the Al-induced shift in H⁺-balance of both genotypes could almost completely be attributed to a decreased NO ₃ ⁻ /NH ₄ ⁺ uptake ratio. In vivo nitrate reductase activity (NRA) was reduced in the shoot of NB9040 and to a lesser degree in SC0283. Al-induced decrease in NRA was accompanied by similar percentual decreases in NO ₃ ⁻ tissue concentrations. Therefore this decrease is interpreted as being indirect,i.e., the consequence of the reduced NO ₃ ⁻ uptake of the plants. A direct repression of NRA by Al seems also unlikely because nitrate reductase activity of the roots (where cellular Al-concentrations should be higher than in shoots) was not affected in Al-treated plants of either genotype.     

DCMU inhibits in vivo nitrate reduction in illuminated barley (C(3)) leaves but not in maize (C(4)): a new mechanism for the role of light?

The leaves of C(4) plants possess a superior metabolic efficiency not only in terms of photosynthetic carbon assimilation, but also in terms of inorganic nitrogen assimilation, when compared to C(3)plants. In vivo nitrate assimilation efficiency of leaves is dependent on light, but the obligatory presence of light has been debated and its role remains confounded. This problem has not been addressed from the standpoint of the C(3) vs. C(4) nature of the species investigated, which may actually hold the key to resolve the controversy. Here, we present the first report providing evidence for differential photo-regulation of leaf nitrate reduction in barley ( Hordeum vulgare L.) vs. maize ( Zea mays L.) plants, which may help explain the superior nitrogen-use efficiency (and hence superior productivity) of maize plants. The novel finding that carbohydrate-depleted maize leaves were able to reduce nitrate when photosynthesis was inhibited by 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) in the presence of light, raises a very important question about the possibilities of a new photo-regulatory mechanism for supporting nitrate reduction in maize leaves operating independently of photosynthetic carbon dioxide fixation. On the other hand, leaves of barley could not carry out any in vivo nitrate assimilation, whatsoever, under these conditions. We find another fundamental difference between the two species in terms of differential regulation of nitrate reductase (NR; EC 1.6.6.1). In barley leaves, NR activity and activation state remained unaffected due to DCMU, but in sharp contrast, both were appreciably upregulated in maize. Collectively, the results indicate that enzyme capacity is not limiting for nitrate reduction in leaves, as the NR activity was higher in barley than in maize. The maize leaves may have had a selective advantage due to C(4) morphology/metabolism in terms of maintaining a better reductant/carbon skeleton supply for nitrate reduction.     

Comparison of bone and osteosarcoma adenylate cyclase. Effects of Mg2+, Ca2+, ATP4? and HATP3? in the assay mixture

The effects of Mg(2+) and Ca(2+) on bone and osteosarcoma adenylate cyclase were investigated. The concentrations of the cations and other ionic species in the assay mixture were calculated by solving the simultaneous equations describing the relevant ionic interactions (multiple equilibria). We re-examined the effects of HATP(3-) and ATP(4-) on enzyme activity and found that (i) the concentration of the minor ATP species is less than 1% of that of MgATP(2-), and their ratio to MgATP(2-) is constant if Mg(2+) and H(+) concentrations are unchanged; (ii) Mg(2+) addition decreased the ratio of the minor species to MgATP(2-) and increased the enzyme activity, but no meaningful kinetic model could attribute this effect of HATP(3-) or ATP(4-). On the other hand, kinetic analysis of Mg(2+) effects showed: (i) stimulation via two metal sites, separate from the catalytic (MgATP(2-)) site, with apparent K(m) values of approximately 1 and 8mm; (ii) that the low affinity increased towards the higher one when the enzyme activity rose as a result of increased substrate or guanine nucleotide concentrations, this effect being less pronounced in tumour; (iii) conversely, that two apparent affinities for MgATP(2-) merged into one at high Mg(2+) concentration; (iv) kinetically, that this relationship is of the mixed con-competitive type, which is consistent with a role for Mg(2+) as a requisite activator, and binding occurring in non-ordered sequence. Analysis of the Ca(2+) effects showed: (i) competition with Mg(2+) at the metal site (K(i) 20mum for bone and 40mum for tumour); (ii) that relative to the substrate the inhibition was uncompetitive, i.e. velocity decreased and affinity increased proportionally, which is consistent with Ca(2+) binding after substrate binding. These findings support the existence of interacting enzyme complexes, losing co-operativity at increased enzyme activity. They also indicate a potential physiological role for Ca(2+) in enzyme regulation and point to quantitative differences between bone and tumour with regard to these properties.     

The Uptake of NO3?, NO2?, and NH4+ by Intact Wheat (Triticum aestivum) Seedlings : I. Induction and Kinetics of Transport Systems

The inducibility and kinetics of the NO₃⁻, NO₂⁻, and NH₄⁺ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for and the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO₃⁻ and NO₂⁻ transporters. Evidence for substrate inducibility of the NH₄⁺ transporters requiring 5 hours is presented. The transport of NO₃⁻ was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO₂⁻ and NH₄⁺. The K_m values for NO₃⁻, NO₂⁻, and NH₄⁺ were, respectively, 0.027, 0.054, and 0.05 millimolar. The K_m for mechanism II of NO₃⁻ transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.     

Importance of L3T4+ and Lyt-2+ cells in the immunologic control of infection with Mycobacterium bovis strain bacillus Calmette-Guérin in mice. Assessment by elimination of T cell subsets in vivo

The course of infection after injection of small doses of bacillus Calmette-Guérin (BCG) was studied in mice which were depleted in vivo of T cell subsets by administration of either anti-L3T4 or anti-Lyt-2 mAb. The results presented herein strongly suggest that the L3T4+ subpopulation play a pivotal role in the immunologic control of BCG infection because the depletion of L3T4+ cells led to a dramatic increase in the number of viable bacteria. Depletion of Lyt-2+ cells had no significant effect on the course of infection. These results were confirmed by using adoptive transfer experiments which showed that protective immunity was mediated by L3T4+ cells generated in the spleen as a result of infection. Moreover, T cells capable of controlling the recurrence of BCG multiplication from residual bacteria remaining in organs after the recovery from infection were shown to belong to the L3T4+ subpopulation.     

Pore water N:P and NH4 +:NO3 ? alter the response of Phragmites australis and Glyceria maxima to extreme nutrient regimes

Phragmites australis and Glyceria maxima are fast-growing littoral grasses often competing for similar wetland habitats. Eutrophication affects their competitiveness, but the outcome is not generally predictable due to the complexity of interrelated factors. We hypotheses that pore water N:P and NH₄ ⁺:NO₃ ^? modify their growth and metabolic responses to the trophic status of the habitat. The hypothesis was tested under standardized conditions of long-term sand cultures. Application of N?+?P up to extreme levels in combination with N:P?<?10 and NH₄ ⁺:NO₃ ^??<?1 triggered positive growth response in both species. In contrast, similar N levels applied in N:P?>?90 and NH₄ ⁺:NO₃ ^??=?4 caused lower productivity, changes in resource allocation, morphology and metabolic relations (e.g. high shoot density, low shoot diameters and heights, reduced root and rhizome growth). Observed signs of stress resembled the factors associated with the reed retreat at the die-back sites. Unbalanced N levels obviously alter plant susceptibility to stresses (altering, e.g. ventilation efficiency, plant anchorage or below-ground storage capacity). The positive effect of sufficient P supply was pronounced in Glyceria. It might therefore favour Glyceria in competition with Phragmites at highly fertile habitats rich in P.     

Organization,replication and modification of the human genome: Temporal order of synthesis and methylation of two classes of HeLa nDNA separated in Ag+−Cs2SO4 gradients

During theHeLa S-phase, DNA was methylated, at 1-hr intervals in isolated nuclei and fractionated in Ag⁺–Cs₂SO₄ gradients providing a heavy GC-rich peak and a main light AT-rich peak. Both size and specific methylation of these peaks changed during the nDNA duplicative phase. Replication of the heavy GC-rich nDNA fraction, which contains genes for ribosomal RNA, occurred inearly S; in contrast, replication of the main AT-rich nDNA fraction was maximal inlate S. Concomitantly, specific methylation of the GC-rich nDNA was maximal in the first part of S, while that of the AT-rich nDNA was maximal in the second part of S. This suggested that genes are replicated and methylated with order during the S-phase.