Human Muscle Protein Synthetic Responses during Weight-Bearing and Non-Weight-Bearing Exercise: A Comparative Study of Exercise Modes and Recovery Nutrition

Effects of conventional endurance (CE) exercise and essential amino acid (EAA) supplementation on protein turnover are well described. Protein turnover responses to weighted endurance exercise (i.e., load carriage, LC) and EAA may differ from CE, because the mechanical forces and contractile properties of LC and CE likely differ. This study examined muscle protein synthesis (MPS) and whole-body protein turnover in response to LC and CE, with and without EAA supplementation, using stable isotope amino acid tracer infusions. Forty adults (mean ± SD, 22 ± 4 y, 80 ± 10 kg, VO_2peak 4.0 ± 0.5 L∙min^-1) were randomly assigned to perform 90 min, absolute intensity-matched (2.2 ± 0.1 VO₂ L∙m^-1) LC (performed on a treadmill wearing a vest equal to 30% of individual body mass, mean ± SD load carried 24 ± 3 kg) or CE (cycle ergometry performed at the same absolute VO₂ as LC) exercise, during which EAA (10 g EAA, 3.6 g leucine) or control (CON, non-nutritive) drinks were consumed. Mixed-muscle and myofibrillar MPS were higher during exercise for LC than CE (mode main effect, P < 0.05), independent of dietary treatment. EAA enhanced mixed-muscle and sarcoplasmic MPS during exercise, regardless of mode (drink main effect, P < 0.05). Mixed-muscle and sarcoplasmic MPS were higher in recovery for LC than CE (mode main effect, P < 0.05). No other differences or interactions (mode x drink) were observed. However, EAA attenuated whole-body protein breakdown, increased amino acid oxidation, and enhanced net protein balance in recovery compared to CON, regardless of exercise mode (P < 0.05). These data show that, although whole-body protein turnover responses to absolute VO₂-matched LC and CE are the same, LC elicited a greater muscle protein synthetic response than CE.     

Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation

Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O is generally attributed to the AT₁ receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose α isoform (PKCα) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O via AT₁ and PKCα-dependent NADPH oxidase activation. In rat TALs, 1 nm Ang II stimulated O from 0.76 ± 0.17 to 1.97 ± 0.21 nmol/min/mg (p < 0.001). An AT₁ antagonist blocked the stimulatory effect of Ang II on O (0.87 ± 0.25 nmol/min/mg; p < 0.006), whereas an AT₂ antagonist had no effect (2.16 ± 0.133 nmol/min/mg; p < 0.05 versus vehicle). Apocynin, an NADPH oxidase inhibitor, blocked Ang II-stimulated O by 90% (p < 0.01). Ang II failed to stimulate O in TALs from p47^phox−/− mice (p < 0.02). Monitored by fluorescence resonance energy transfer, Ang II increased PKC activity from 0.02 ± 0.03 to 0.13 ± 0.02 arbitrary units (p < 0.03). A general PKC inhibitor, GF109203X, blocked the effect of Ang II on O (1.47 ± 0.21 versus 2.72 ± 0.47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and β-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O (0.59 ± 0.15 versus 2.05 ± 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKCα and PKCβ, we used tubules expressing dominant-negative PKCα or -β. In control TALs, Ang II stimulated O by 2.17 ± 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKCα, Ang II failed to stimulate O (change: −0.30 ± 0.27 nmol/min/mg). In tubules expressing dominant-negative PKCβ1, Ang II stimulated O by 2.08 ± 0.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O production via activation of AT₁ receptors and PKCα-dependent NADPH oxidase.     

Immobilised Histidine Tagged β 2-Adrenoceptor Oriented by a Diazonium Salt Reaction and Its Application in Exploring Drug-Protein Interaction Using Ephedrine and Pseudoephedrine as Probes

A new oriented method using a diazonium salt reaction was developed for linking β ₂-adrenoceptor (β ₂-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β ₂-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β ₂-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×10³/M for ephedrine and (3.80±0.02) ×10³/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10⁻⁴ M. Thermodynamic studies showed that the binding of the two compounds to β ₂-AR was a spontaneous reaction with exothermal processes. The ΔG^θ, ΔH^θ and ΔS^θ for the interaction between ephedrine and β ₂-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β ₂-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.     

Crystal Structure,Cytotoxicity and Interaction with DNA of Zinc (II) Complexes with o-Vanillin Schiff Base Ligands

Two new zinc complexes, Zn(HL¹)₂ (1) and [Zn₂(H₂L²)(OAc)₂]₂ (2) [H₂L¹ = Schiff base derived from o-vanillin and (R)-(+)-2-amino-3-phenyl-1-propanol, H₃L² = Schiff base derived from o-vanillin and 2-amino-2-ethyl-1,3-propanediol], have been synthesized and characterized by single crystal X-ray diffraction, elemental analyses, TG analyses, solid fluorescence, IR, UV-Vis and circular dichroism spectra. The structural analysis shows that complex 1 has a right-handed double helical chain along the crystallographic b axis. A homochiral 3D supramolecular architecture has been further constructed by intermolecular C-H··· π, O-H···O and C-H···O interactions. Complex 2 includes two crystallographically independent binuclear zinc molecules. The two binuclear zinc molecules are isostructural. The 2-D sheet supramolecular structure was formed by intermolecular hydrogen bonding interaction. The fluorescence of ligands and complexes in DMF at room temperature are studied. The interactions of two complexes with calf thymus DNA (CT-DNA) are investigated using UV-Vis, CD and fluorescence spectroscopy. The results show that complex 1 exhibits higher interaction with CT-DNA than complex 2. In addition, in vitro cytotoxicity of the complexes towards four kinds of cancerous cell lines (A549, HeLa, HL-60 and K562) were assayed by the MTT method. Investigations on the structures indicated that the chirality and nuclearity of zinc complexes play an important role on cytotoxic activity.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

CO2-induced ion and fluid transport in human retinal pigment epithelium

In the intact eye, the transition from light to dark alters pH, [Ca²⁺], and [K] in the subretinal space (SRS) separating the photoreceptor outer segments and the apical membrane of the retinal pigment epithelium (RPE). In addition to these changes, oxygen consumption in the retina increases with a concomitant release of CO₂ and H₂O into the SRS. The RPE maintains SRS pH and volume homeostasis by transporting these metabolic byproducts to the choroidal blood supply. In vitro, we mimicked the transition from light to dark by increasing apical bath CO₂ from 5 to 13%; this maneuver decreased cell pH from 7.37 ± 0.05 to 7.14 ± 0.06 (n = 13). Our analysis of native and cultured fetal human RPE shows that the apical membrane is significantly more permeable (≈10-fold; n = 7) to CO₂ than the basolateral membrane, perhaps due to its larger exposed surface area. The limited CO₂ diffusion at the basolateral membrane promotes carbonic anhydrase–mediated HCO₃ transport by a basolateral membrane Na/nHCO₃ cotransporter. The activity of this transporter was increased by elevating apical bath CO₂ and was reduced by dorzolamide. Increasing apical bath CO₂ also increased intracellular Na from 15.7 ± 3.3 to 24.0 ± 5.3 mM (n = 6; P < 0.05) by increasing apical membrane Na uptake. The CO₂-induced acidification also inhibited the basolateral membrane Cl/HCO₃ exchanger and increased net steady-state fluid absorption from 2.8 ± 1.6 to 6.7 ± 2.3 µl × cm⁻² × hr⁻¹ (n = 5; P < 0.05). The present experiments show how the RPE can accommodate the increased retinal production of CO₂ and H₂O in the dark, thus preventing acidosis in the SRS. This homeostatic process would preserve the close anatomical relationship between photoreceptor outer segments and RPE in the dark and light, thus protecting the health of the photoreceptors.     

Water transport in the midrib tissue of maize leaves : direct measurement of the propagation of changes in cell turgor across a plant tissue

Water movement across plant tissues occurs along two paths: from cell-to-cell and in the apoplasm. We examined the contribution of these two paths to the kinetics of water transport across the parenchymatous midrib tissue of the maize (Zea mays L.) leaf. Water relations parameters (hydraulic conductivity, Lp; cell elastic coefficient, ε; half-time of water exchange for individual cells, T_½) of individual parenchyma cells determined with the pressure probe varied in different regions of the midrib. In the adaxial region, Lp = (0.3 ± 0.3)·10⁻⁵ centimeters per second per bar, ε = 103 ± 72 bar, and T_½ = 7.9 ± 4.8 seconds (n = seven cells); whereas, in the abaxial region, Lp = (2.5 ± 0.9)·10⁻⁵ centimeters per second per bar, ε = 41 ± 9 bar, and T_½ = 1.3 ± 0.5 seconds (n = 7). This zonal variation in Lp, ε, and T_½ indicates that tissue inhomogeneities exist for these parameters and could have an effect on the kinetics of water transport across the tissue.

The diffusivity of the tissue to water (D_t) obtained from the sorption kinetics of rehydrating tissue was D_t = (1.1 ± 0.4)·10⁻⁶ square centimeters per second (n = 6). The diffusivity of the cell-to-cell path (D_c) calculated from pressure probe data ranged from D_c = 0.4·10⁻⁶ square centimeters per second in the adaxial region to D_c = 6.1·10⁻⁶ square centimeters per second in the abaxial region of the tissue. D_t D_c suggests substantial cell-to-cell transport of water occurred during rehydration. However, the tissue diffusivity calculated from the kinetics of pressure-propagation across the tissue (D_t′) was D_t′ = (33.1 ± 8.0)·10⁻⁶ square centimeters per second (n = 8) and more than 1 order of magnitude larger than D_t. Also, the hydraulic conductance of the midrib tissue (Lp_m per square centimeter of surface) estimated from pressure-induced flows across several parenchyma cell layers was Lp_m = (8.9 ± 5.6)·10⁻⁵ centimeters per second per bar (n = 5) and much larger than Lp.

These results indicate that the preferential path for water transport across the midrib tissue depends on the nature of the driving forces present within the tissue. Under osmotic conditions, the cell-to-cell path dominates, whereas under hydrostatic conditions water moves primarily in the apoplasm.

     

Isolation and Characterization of Carbendazim-degrading Rhodococcus erythropolis djl-11

Carbendazim (methyl 1H-benzimidazol-2-yl carbamate) is one of the most widely used fungicides in agriculture worldwide, but has been reported to have adverse effects on animal health and ecosystem function. A highly efficient carbendazim-degrading bacterium (strain dj1-11) was isolated from carbendazim-contaminated soil samples via enrichment culture. Strain dj1-11 was identified as Rhodococcus erythropolis based on morphological, physiological and biochemical characters, including sequence analysis of the 16S rRNA gene. In vitro degradation of carbendazim (1000 mg·L⁻¹) by dj1-11 in minimal salts medium (MSM) was highly efficient, and with an average degradation rate of 333.33 mg·L⁻¹·d⁻¹ at 28°C. The optimal temperature range for carbendazim degradation by dj1-11 in MSM was 25–30°C. Whilst strain dj1-11 was capable of metabolizing cabendazim as the sole source of carbon and nitrogen, degradation was significantly (P<0.05) increased by addition of 12.5 mM NH₄NO₃. Changes in MSM pH (4–9), substitution of NH₄NO₃ with organic substrates as N and C sources or replacing Mg²⁺ with Mn²⁺, Zn²⁺ or Fe²⁺ did not significantly affect carbendazim degradation by dj1-11. During the degradation process, liquid chromatography-mass spectrometry (LC-MS) detected the metabolites 2-aminobenzimidazole and 2-hydroxybenzimidazole. A putative carbendazim-hydrolyzing esterase gene was cloned from chromosomal DNA of djl-11 and showed 99% sequence homology to the mheI carbendazim-hydrolyzing esterase gene from Nocardioides sp. SG-4G.     

Binding Rather Than Metabolism May Explain the Interaction of Two Food-Grade Lactobacillus Strains with Zearalenone and Its Derivative ά-Zearalenol

The interaction between two Fusarium mycotoxins, zearalenone (ZEN) and its derivative ¯α-zearalenol (¯α-ZOL), with two food-grade strains of Lactobacillus was investigated. The mycotoxins (2 μg ml⁻¹) were incubated with either Lactobacillus rhamnosus strain GG or L. rhamnosus strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and ¯α-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or ¯α-ZOL by lyophilized L. rhamnosus GG and L. rhamnosus LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with ¯α-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

Influences of Land Use/Cover Types on Nitrous Oxide Emissions during Freeze-Thaw Periods from Waterlogged Soils in Inner Mongolia

Nitrous oxide emissions during freeze/thaw periods contribute significantly to annual soil N₂O emissions budgets in middle- and high-latitude areas; however, the freeze/thaw-related N₂O emissions from waterlogged soils have hardly been studied in the Hulunber Grassland, Inner Mongolia. For this study, the effects of changes in land use/cover types on N₂O emissions during freeze–thaw cycles were investigated to more accurately quantify the annual N₂O emissions from grasslands. Soil cores from six sites were incubated at varying temperature (ranging from −15 to 10°C) to simulate freeze–thaw cycles. N₂O production rates were low in all soil cores during freezing periods, but increased markedly after soil thawed. Mean rates of N₂O production differed by vegetation type, and followed the sequence: Leymus chinensis (LC) and Artemisia tanacetifolia (AT) steppes > LC steppes ≥ Stipa baicalensis (SB) steppes. Land use types (mowing and grazing) had differing effects on freeze/thaw-related N₂O production. Grazing significantly reduced N₂O production by 36.8%, while mowing enhanced production. The production of N₂O was related to the rate at which grassland was mowed, in the order: triennially (M3) > once annually (M1) ≥ unmown (UM). Compared with the UM control plot, the M3 and M1 mowing regimes enhanced N₂O production by 57.9% and 13.0% respectively. The results of in situ year-round measurements showed that large amounts of N₂O were emitted during the freeze–thaw period, and that annual mean fluxes of N₂O were 9.21 μg N₂O-N m^-2 h^-1 (ungrazed steppe) and 6.54 μg N₂O-N m^-2 h^-1 (grazed steppe). Our results further the understanding of freeze/thaw events as enhancing N₂O production, and confirm that different land use/cover types should be differentiated rather than presumed to be equivalent, regarding nitrous oxide emission. Even so, further research involving multi-year and intensive measurements of N₂O emission is still needed.     

Filtration Coefficient of the Axon Membrane As Measured with Hydrostatic and Osmotic Methods

The hydraulic conductivity of the membranes surrounding the giant axon of the squid, Dosidicus gigas, was measured. In some axons the axoplasm was partially removed by suction. Perfusion was then established by insertion of a second pipette. In other axons the axoplasm was left intact and only one pipette was inserted. In both groups hydrostatic pressure was applied by means of a water column in a capillary manometer. Displacement of the meniscus in time gave the rate of fluid flowing across the axon sheath. In both groups osmotic differences across the membrane were established by the addition of a test molecule to the external medium which was seawater. The hydraulic conductivity determined by application of hydrostatic pressure was 10.6 ± 0.8.10^-8 cm/sec cm H₂O in perfused axons and 3.2 ± 0.6.10^-8 cm/sec cm H₂O in intact axons. When the driving force was an osmotic pressure gradient the conductivity was 4.5 ± 0.6 x 10^-10 cm/sec cm H₂O and 4.8 ± 0.9 x 10^-10 cm/sec cm H₂O in perfused and intact axons, respectively. A comparable result was found when the internal solution was made hyperosmotic. The fluid flow was a linear function of the hydrostatic pressure up to 70 cm of water. Glycerol outflux and membrane conductance were increased 1.6 and 1.1 times by the application of hydrostatic pressure. These increments do not give an explanation of the difference between the filtration coefficients. Other possible explanations are suggested and discussed.     

In-depth analysis of N2O fluxes in tropical forest soils of the Congo Basin combining isotope and functional gene analysis

Primary tropical forests generally exhibit large gaseous nitrogen (N) losses, occurring as nitric oxide (NO), nitrous oxide (N₂O) or elemental nitrogen (N₂). The release of N₂O is of particular concern due to its high global warming potential and destruction of stratospheric ozone. Tropical forest soils are predicted to be among the largest natural sources of N₂O; however, despite being the world’s second-largest rainforest, measurements of gaseous N-losses from forest soils of the Congo Basin are scarce. In addition, long-term studies investigating N₂O fluxes from different forest ecosystem types (lowland and montane forests) are scarce. In this study we show that fluxes measured in the Congo Basin were lower than fluxes measured in the Neotropics, and in the tropical forests of Australia and South East Asia. In addition, we show that despite different climatic conditions, average annual N₂O fluxes in the Congo Basin’s lowland forests (0.97 ± 0.53 kg N ha⁻¹ year⁻¹) were comparable to those in its montane forest (0.88 ± 0.97 kg N ha⁻¹ year⁻¹). Measurements of soil pore air N₂O isotope data at multiple depths suggests that a microbial reduction of N₂O to N₂ within the soil may account for the observed low surface N₂O fluxes and low soil pore N₂O concentrations. The potential for microbial reduction is corroborated by a significant abundance and expression of the gene nosZ in soil samples from both study sites. Although isotopic and functional gene analyses indicate an enzymatic potential for complete denitrification, combined gaseous N-losses (N₂O, N₂) are unlikely to account for the missing N-sink in these forests. Other N-losses such as NO, N₂ via Feammox or hydrological particulate organic nitrogen export could play an important role in soils of the Congo Basin and should be the focus of future research.Subject terms: Microbiology, Biogeochemistry     

Rhizoremediation of Trichloroethylene by a Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-Monooxygenase Constitutively

Trichloroethylene (TCE) was removed from soils by using a wheat rhizosphere established by coating seeds with a recombinant, TCE-degrading Pseudomonas fluorescens strain that expresses the tomA⁺ (toluene o-monooxygenase) genes from Burkholderia cepacia PR1₂₃(TOM_23C). A transposon integration vector was used to insert tomA⁺ into the chromosome of P. fluorescens 2-79, producing a stable strain that expressed constitutively the monooxygenase at a level of 1.1 nmol/min · mg of protein (initial TCE concentration, 10 μM, assuming that all of the TCE was in the liquid) for more than 280 cell generations (36 days). We also constructed a salicylate-inducible P. fluorescens strain that degraded TCE at an initial rate of 2.6 nmol/min · mg of protein in the presence of 10 μM TCE [cf. B. cepacia G4 PR1₂₃(TOM_23C), which degraded TCE at an initial rate of 2.5 nmol/min · mg of protein]. A constitutive strain, P. fluorescens 2-79TOM, grew (maximum specific growth rate, 0.78 h⁻¹) and colonized wheat (3 × 10⁶ CFU/cm of root) as well as wild-type P. fluorescens 2-79 (maximum specific growth rate, 0.77 h⁻¹; level of colonization, 4 × 10⁶ CFU/cm of root). Rhizoremediation of TCE was demonstrated by using microcosms containing the constitutive monooxygenase-expressing microorganism, soil, and wheat. These closed microcosms degraded an average of 63% of the initial TCE in 4 days (20.6 nmol of TCE/day · plant), compared to the 9% of the initial TCE removed by negative controls consisting of microcosms containing wild-type P. fluorescens 2-79-inoculated wheat, uninoculated wheat, or sterile soil.     

Reduction of the Powerful Greenhouse Gas N2O in the South-Eastern Indian Ocean

Nitrous oxide (N₂O) is a powerful greenhouse gas and a key catalyst of stratospheric ozone depletion. Yet, little data exist about the sink and source terms of the production and reduction of N₂O outside the well-known oxygen minimum zones (OMZ). Here we show the presence of functional marker genes for the reduction of N₂O in the last step of the denitrification process (nitrous oxide reductase genes; nosZ) in oxygenated surface waters (180–250 O₂ μmol.kg^-1) in the south-eastern Indian Ocean. Overall copy numbers indicated that nosZ genes represented a significant proportion of the microbial community, which is unexpected in these oxygenated waters. Our data show strong temperature sensitivity for nosZ genes and reaction rates along a vast latitudinal gradient (32°S-12°S). These data suggest a large N₂O sink in the warmer Tropical waters of the south-eastern Indian Ocean. Clone sequencing from PCR products revealed that most denitrification genes belonged to Rhodobacteraceae. Our work highlights the need to investigate the feedback and tight linkages between nitrification and denitrification (both sources of N₂O, but the latter also a source of bioavailable N losses) in the understudied yet strategic Indian Ocean and other oligotrophic systems.     

Coarsening Dynamics of Domains in Lipid Membranes

We investigate isothermal diffusion and growth of micron-scale liquid domains within membranes of free-floating giant unilamellar vesicles with diameters between 80 and 250 μm. Domains appear after a rapid temperature quench, when the membrane is cooled through a miscibility phase transition such that coexisting liquid phases form. In membranes quenched far from a miscibility critical point, circular domains nucleate and then progress within seconds to late stage coarsening in which domains grow via two mechanisms 1), collision and coalescence of liquid domains, and 2), Ostwald ripening. Both mechanisms are expected to yield the same growth exponent, α = 1/3, where domain radius grows as time^α. We measure α = 0.28 ± 0.05, in excellent agreement. In membranes close to a miscibility critical point, the two liquid phases in the membrane are bicontinuous. A quench near the critical composition results in rapid changes in morphology of elongated domains. In this case, we measure α = 0.50 ± 0.16, consistent with theory and simulation.     

Laser activation of rapid absorption changes in spinach chloroplasts and chlorella

The kinetics of the 520 mμ absorption change in spinach chloroplasts and Chlorella vulgaris following a flash from the ruby laser have been determined as follows: rise halftime ≤ 0.3 × 10⁻⁶ second; rapid recovery halftime = 5 to 6 × 10⁻⁶ second; intermediate recovery halftime = 4 × 10⁻⁴ second (spinach chloroplasts only); slow recovery halftime = 12 to 170 × 10⁻³ second, dependent on the measuring light intensity and aerobicity of the suspension.

The rapid phase of the 520 mμ reaction is approximately independent of temperature, from 295° to 77° Absolute.

With increasing oxygenation of the sample, the extent of the rapid phase decreases, the extent of the slow phase increases, while the extent of the intermediate phase in spinach chloroplasts remains constant.

In spinach chloroplasts, no recovery halftime of the 3 recovery phases for the 520 mμ absorption change was observed to correspond to the halftime for oxidation of cytochrome f (t_½ = 1.3 × 10⁻³ second).

     

On the Relation between Effector Concentration and the Rate of Induced Enzyme Synthesis

The Jacob and Monod scheme for the regulation of enzyme formation leads to the following relation between the relative rate of enzyme synthesis α and cellular effector concentration E (the lower sign is for repressible systems): log (α/1 - α - α_b) = ± n log [E] + log α_b ± log K₁. This equation permits linear plotting of experimental data and the evaluation of three quantities: n, the number of effector molecules combining with a repressor molecule, K₁, the dissociation constant of this interaction and K₂/R_t, the ratio of repressor-operator dissociation constant to total repressor concentration. Measurements on the repression of alkaline phosphatase in Escherichia coli as a function of phosphate concentration are reported and fit the proposed equation with n = 1, indicating that the binding of a single phosphate to the repressor species may be sufficient to cause repression. K₁ of this interaction was found to be 0.58 ±0.11 × 10^-3 M. The available data regarding the enzymes of the lac operon in a variety of E. coli strains, and several other enzymes are analyzed. It is confirmed that the lac repressor interacts with 2 isopropyl thiogalactoside (IPTG) molecules to relieve repression with a K₁ = 50 ±20 × 10^-12 M². In some strains, separate binding constants for the first and second IPTG molecules can be evaluated.     

A study of lipid bilayer membrane stability using precise measurements of specific capacitance

A method is described for measuring the specific capacitance (C_m) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10^-2 M KCl show that C_m depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), C_m increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm² with a time constant of ~15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. C_m of final-state membranes depends upon applied voltage (V_a) and obeys the equation C_m = C₀ + βV_a² where V_a V_DC + V^rms_AC. C₀ and β depend upon temperature; C₀ decreases linearly with temperature while β increases linearly. At 20°C, C₀ = 0.559 ±0.01 (SD) μF/cm² and β = 0.0123 ±0.0036 (SD) (μF/cm²)/(mv²) and at 34°C, C₀ = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in C_m are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves.