An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO₂⁻) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO₃⁻) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO₃⁻→NO₂⁻→NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O₂, NO₂⁻, and NO₃⁻ in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO₃⁻, which was relieved upon O₂ depletion. As cell growth depleted dissolved O₂ levels, NO₃⁻ was converted to NO₂⁻ at near-stoichiometric levels, whereas NO₂⁻ consumption did not coincide with NO or NO₃⁻ accumulation. Assimilatory NO₂⁻ reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.     

Synthesis and anti-inflammatory activity of celecoxib like compounds

Nine novel 4-[3-(4-Dimethylamino-phenyl)-5-aryl-4,5-dihydro-pyrazol-1-yl]-benzenesulfonamides (2a-i) were synthesized and evaluated for their anti-inflammatory and antiproliferative activities. These compounds (2a-i) showed moderate to strong anti-inflammatory activity in carrageenan rat paw oedema test. Compounds 2b, 2d and 2g showing comparable anti-inflammatory activity to that of reference drug celecoxib were evaluated for their ulcerogenic and analgesic activities. The effect of 2b, 2d and 2g on the content of NO, TNF-α and PGE₂ in exudates from rat paw stimulated by carrageenan was also evaluated. The compound 2c showed considerable antitumor activities against all 60 human tumor cell lines with effective GI₅₀ (MG-MID) value of 3.63 µM. It exhibited maximum activity against melanoma (LOX IMVI and SK-MEL-5) cancer cell lines with GI₅₀ value less than 2 μM.     

7. Global support and coordination: conserving germplasm of world crop species and their relatives

The awareness of the importance of plant biodiversity has been considerably raised in both developed and developing countries over the last decade. Some of the debate has not been helpful in fostering collaboration or progress towards a more rational support network. The paper attempts to identify and categorize existing efforts in operation in a wide range of institutions and bodies ranging from essentially environmentally orientated to crop-based organizations. Current funding and training initiatives are discussed.     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Diagnostic parameters for selecting against novel spruce (Picea abies) decline: IV. Response of photosynthesis and transpiration to SO2+NO2 exposures

The dose- and time-response effects of 3 days of 6 h day-time sequential exposures to NO₂, SO₂ and SO₂+NO₂ of 0.45–1.81 μl l⁻¹ (ppm) SO₂ and 1.50–7.65 μl l⁻¹ NO₂ on photosynthesis, transpiration and dark respiration were examined for nine Carpatho-Ukrainian half-sib families and a population from the GFR ('Westerhof') of Norway spruce [ Piecea abies (L.) Karst.], all in their 5th growing season.
SO₂+NO₂ inhibited photosynthesis and transpiration and stimulated dark respiration more than SO₂ alone. SO₂ and SO₂+NO₂ at the lowest concentrations inhibited night transpiration, but increased it at the highest concentration, the strongest effects being obtained with combined exposures. Photosynthesis of the different half-sib families was affected significantly differently by SO₂+NO₂ exposures. NO₂ alone had no effects.
Sensitivity to transpiration decline correlated negatively with branch density. Height of trees correlated postitively with decline sensitivity in the seed orchard. The distribution of photosynthesis and transpiration sensitivities over all tested half-sib families correlated negatively with the distribution of decline sensitivity of their parents in a rural Danish seed orchard. The relative photosynthesis and transpiration sensitivities may thus serve as diagnostic parameters for selecting against novel spruce decline.     

Influence of partial defoliation of green pepper on the senescence,growth, and nitrate reductase of the remaining leaf

Summary Removal of all but one leaf from pepper plants prevented the senescence of the remaining leaf and caused increases of approximately 140, 200, and 200%, respectively in leaf area, weight, and nitrate reductase activity. Development of the fruit (fresh and dry weight increases) was only approximately 65% of that of fruit on control plants.     

Oxidation-reduction properties of the electron acceptors of Photosystem II I. Redox titration of the flash-induced carotenoid band shift,of C550 and of the variable fluorescence yield in spinach chloroplasts

Redox titration of the electrochromic carotenoid band shift, detected at 50 μs after a saturating actinic flash, in spinach chloroplasts, shows that only one electron acceptor in Photosystem II participates in a transmembrane primary electron transfer. This species, the primary quinone acceptor, Q, shows only one midpoint potential (E_m,7.5) of approx. 0 V and is undoubtedly equivalent to the fluorescence quencher, Q_H. A second titration wave is observed at low potential ($\text{E}{}_{\mathrm{<mtext>m</mtext><mtext>,7.5</mtext>}}\text{? ? 240}\text{mV}$) and at greater than 3 ms after a saturating actinic flash. This wave has an action spectrum different from that of Photosystem II centers containing Q and could arise from a secondary but not primary electron transfer. A low-potential fluorescence quencher is observed in chloroplasts which largely disappears in a single saturating flash at ? 185 mV and which does not participate in a transmembrane electron transfer. This low-potential quencher (probably equivalent to fluorescence quencher, Q_L) and Q are altogether different species. Redox titration of C550 shows that if electron acceptor Q_β is indeed characterized by an E_m,7 of + 120 mV, then this acceptor does not give rise to a C550 signal upon reduction and does not participate in a transmembrane electron transfer. This titration also shows that C550 is not associated with Q_L.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Energetics of cell-cell and cell-biopolymer interactions

The energy vs distance balance of cell suspensions (in the presence and in the absence of extracellular biopolymer solutions) is studied, not only in the light of the classical Derjaguin-Landau-Verwey-Over-beek (DLVO) theory (which considered just the electrostatic (EL) and Lifshitz-van der Waals (LW) interactions), but also by taking electron-acceptor/electron-donor, or Lewis acid-base (AB) and osmotic (OS) interactions into account. Since cell surfaces, as well as many biopolymers tend to have strong monopolar electron-donor properties, they are able to engage in a strong mutual AB repulsion when immersed in a polar liquid such as water. The effects of that repulsion have been observed earlier in the guise of hydration pressure. The AB repulsion is, at close range, typically one or two orders of magnitude stronger than the EL repulsion, but its rate of decay is much steeper. In most cases, AB interactions are quantitatively the dominant factor in cell stability (when repulsive) and in “hydrophobic interactions” (when attractive). OS interactions exerted by extracellularly dissolved biopolymers are weak, but their rate of decay is very gradual, so OS repulsions engendered by biopolymer solutions may be of importance in certain long-range interactions. OS interactions exerted by biopolymers attached to cells or particles (e.g., by glycocalix glycoproteins), are very short-ranged and usually are negligibly small in comparison with the other interaction forces, in aqueous media.