Nitrate Reductase Activity in Soybeans (Glycine max [L.] Merr.): II. Energy Limitations

Growth chamber studies with soybeans (Glycine max [L.] Merr.) were designed to determine the relative limitations of NO₃⁻, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO₃⁻in vivo, −NO₃⁻in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO₃⁻ to the in vivo NR assay medium increased activity (over that of the −NO₃⁻in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO₃⁻ was rate-limiting. The stimulation of in vivo NR activity by NO₃⁻ was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO₃⁻ was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO₂⁻ [g fresh weight, hr]⁻¹) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.     

The CoxD Protein,a Novel AAA+ ATPase Involved in Metal Cluster Assembly: Hydrolysis of Nucleotide-Triphosphates and Oligomerization

CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO₂] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk), preparations of CoxD revealed a mean K_M value of 0.69±0.14 mM ATP and an apparent V_max value of 19.3±2.3 nmol ATP hydrolyzed min⁻¹ mg⁻¹. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer), preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10–16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO₂] cluster assembly are discussed.     

Nitrogen, Carbon, and Sulfur Metabolism in Natural Thioploca Samples

Filamentous sulfur bacteria of the genus Thioploca occur as dense mats on the continental shelf off the coast of Chile and Peru. Since little is known about their nitrogen, sulfur, and carbon metabolism, this study was undertaken to investigate their (eco)physiology. Thioploca is able to store internally high concentrations of sulfur globules and nitrate. It has been previously hypothesized that these large vacuolated bacteria can oxidize sulfide by reducing their internally stored nitrate. We examined this nitrate reduction by incubation experiments of washed Thioploca sheaths with trichomes in combination with ¹⁵N compounds and mass spectrometry and found that these Thioploca samples produce ammonium at a rate of 1 nmol min⁻¹ mg of protein⁻¹. Controls showed no significant activity. Sulfate was shown to be the end product of sulfide oxidation and was observed at a rate of 2 to 3 nmol min⁻¹ mg of protein⁻¹. The ammonium and sulfate production rates were not influenced by the addition of sulfide, suggesting that sulfide is first oxidized to elemental sulfur, and in a second independent step elemental sulfur is oxidized to sulfate. The average sulfide oxidation rate measured was 5 nmol min⁻¹ mg of protein⁻¹ and could be increased to 10.7 nmol min⁻¹ mg of protein⁻¹ after the trichomes were starved for 45 h. Incorporation of ¹⁴CO₂ was at a rate of 0.4 to 0.8 nmol min⁻¹ mg of protein⁻¹, which is half the rate calculated from sulfide oxidation. [2-¹⁴C]acetate incorporation was 0.4 nmol min⁻¹ mg of protein⁻¹, which is equal to the CO₂ fixation rate, and no ¹⁴CO₂ production was detected. These results suggest that Thioploca species are facultative chemolithoautotrophs capable of mixotrophic growth. Microautoradiography confirmed that Thioploca cells assimilated the majority of the radiocarbon from [2-¹⁴C]acetate, with only a minor contribution by epibiontic bacteria present in the samples.     

Deletion of Ubiquitin Fold Modifier Protein Ufm1 Processing Peptidase Ufsp in L. donovani Abolishes Ufm1 Processing and Alters Pathogenesis

Previously, we showed Leishmania donovani Ufm1 has a Gly residue conserved at the C-terminal region with a unique 17 amino acid residue extension that must be processed prior to conjugation to target proteins. In this report, we describe for the first time the isolation and characterization of the Leishmania Ufm1-specific protease Ufsp. Biochemical analysis of L. donovani Ufsp showed that this protein possesses the Ufm1 processing activity using sensitive FRET based activity probes. The Ufm1 cleavage activity was absent in a mutant Ufsp in which the active site cysteine is altered to a serine. To examine the effects of abolition of Ufm1 processing activity, we generated a L. donovani null mutant of Ufsp (LdUfsp^−/−). Ufm1 processing activity was abolished in LdUfsp^−/− mutant, and the processing defect was reversed by re-expression of wild type but not the cys>ser mutant in the LdUfsp^−/− parasites. Further LdUfsp^−/− mutants showed reduced survival as amastigotes in infected human macrophages but not as promastigotes. This growth defect in the amastigotes was reversed by re-expression of wild type but not the cys>ser mutant in the Ufsp^−/− indicating the essential nature of this protease for Leishmania pathogenesis. Further, mouse infection experiments showed deletion of Ufsp results in reduced virulence of the parasites. Additionally, Ufsp activity was inhibited by an anti-leishmanial drug Amphotericin B. These studies provide an opportunity to test LdUfsp^−/− parasites as drug and vaccine targets.     

Ae4 (Slc4a9) Anion Exchanger Drives Cl? Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

Transcellular Cl⁻ movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na⁺-K⁺-2Cl⁻ cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl⁻ above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl⁻ uptake pathway concentrates Cl⁻ ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl⁻/HCO₃⁻ exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2^−/− mice. In contrast, saliva secretion was reduced by 35% in Ae4^−/− mice. The decrease in salivation was not related to loss of Na⁺-K⁺-2Cl⁻ cotransporter or Na⁺/H⁺ exchanger activity in Ae4^−/− mice but correlated with reduced Cl⁻ uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl⁻/HCO₃⁻ exchanger activity revealed that HCO₃⁻-dependent Cl⁻ uptake was reduced in the acinar cells of Ae2^−/− and Ae4^−/− mice. Moreover, Cl⁻/HCO₃⁻ exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl⁻/HCO₃⁻ exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.     

Kinetic Parameters of the Conversion of Methane Precursors to Methane in a Hypereutrophic Lake Sediment

The kinetic parameters K_m, V_max, T_t (turnover time), and v (natural velocity) were determined for H₂ and acetate conversion to methane by Wintergreen Lake sediment, using short-term (a few hours) methods and incubation temperatures of 10 to 14°C. Estimates of the Michaelis-Menten constant, K_m, for both the consumption of hydrogen and the conversion of hydrogen to methane by sediment microflora averaged about 0.024 μmol g⁻¹ of dry sediment. The maximal velocity, V_max, averaged 4.8 μmol of H₂ g⁻¹ h⁻¹ for hydrogen consumption and 0.64 μmol of CH₄ g⁻¹ h⁻¹ for the conversion of hydrogen to methane during the winter. Estimated natural rates of hydrogen consumption and hydrogen conversion to methane could be calculated from the Michaelis-Menten equation and estimates of K_m, V_max, and the in situ dissolved-hydrogen concentration. These results indicate that methane may not be the only fate of hydrogen in the sediment. Among several potential hydrogen donors tested, only formate stimulated the rate of sediment methanogenesis. Formate conversion to methane was so rapid that an accurate estimate of kinetic parameters was not possible. Kinetic experiments using [2-¹⁴C]acetate and sediments collected in the summer indicated that acetate was being converted to methane at or near the maximal rate. A minimum natural rate of acetate conversion to methane was estimated to be about 110 nmol of CH₄ g⁻¹ h⁻¹, which was 66% of the V_max (163 nmol of CH₄ g⁻¹ h⁻¹). A 15-min preincubation of sediment with 5.0 × 10⁻³ atm of hydrogen had a pronounced effect on the kinetic parameters for the conversion of acetate to methane. The acetate pool size, expressed as the term K_m + S_n (S_n is in situ substrate concentration), decreased by 37% and T_t decreased by 43%. The V_max remained relatively constant. A preincubation with hydrogen also caused a 37% decrease in the amount of labeled carbon dioxide produced from the metabolism of [U-¹⁴C]valine by sediment heterotrophs.     

3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation

A modified 3-hydroxypropionate cycle has been proposed as the autotrophic CO₂ fixation pathway for the thermoacidophilic crenarchaeon Metallosphaera sedula. The cycle requires the reductive conversion of 3-hydroxypropionate to propionyl-coenzyme A (propionyl-CoA). The specific activity of the 3-hydroxypropionate-, CoA-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.023 μmol min⁻¹mg protein⁻¹. The reaction sequence is catalyzed by at least two enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the following reaction: 3-hydroxypropionate + ATP + CoA → 3-hydroxypropionyl-CoA + AMP + PP_i. The enzyme was purified 95-fold to a specific activity of 18 μmol min⁻¹ mg protein⁻¹ from autotrophically grown M. sedula cells. An internal peptide sequence was determined and a gene encoding a homologous protein identified in the genome of Sulfolobus tokodaii; similar genes were found in S. solfataricus and S. acidocaldarius. The gene was heterologously expressed in Escherichia coli, and the His-tagged protein was purified. Both the native enzyme from M. sedula and the recombinant enzyme from S. tokodaii not only activated 3-hydroxypropionate to its CoA ester but also activated propionate, acrylate, acetate, and butyrate; however, with the exception of propionate, the affinities for these substrates were reduced. 3-Hydroxypropionyl-CoA synthetase is up-regulated eightfold in autotrophically versus heterotrophically grown M. sedula, supporting its proposed role during CO₂ fixation in this archaeon and possibly other members of the Sulfolobaceae family.     

Binding of Cysteine Synthase to the STAS Domain of Sulfate Transporter and Its Regulatory Consequences

The sulfate ion (SO₄²⁻) is transported into plant root cells by SO₄²⁻ transporters and then mostly reduced to sulfide (S²⁻). The S²⁻ is then bonded to O-acetylserine through the activity of cysteine synthase (O-acetylserine (thiol)lyase or OASTL) to form cysteine, the first organic molecule of the SO₄²⁻ assimilation pathway. Here, we show that a root plasma membrane SO₄²⁻ transporter of Arabidopsis, SULTR1;2, physically interacts with OASTL. The interaction was initially demonstrated using a yeast two-hybrid system and corroborated by both in vivo and in vitro binding assays. The domain of SULTR1;2 shown to be important for association with OASTL is called the STAS domain. This domain is at the C terminus of the transporter and extends from the plasma membrane into the cytoplasm. The functional relevance of the OASTL-STAS interaction was investigated using yeast mutant cells devoid of endogenous SO₄²⁻ uptake activity but co-expressing SULTR1;2 and OASTL. The analysis of SO₄²⁻ transport in these cells suggests that the binding of OASTL to the STAS domain in this heterologous system negatively impacts transporter activity. In contrast, the activity of purified OASTL measured in vitro was enhanced by co-incubation with the STAS domain of SULTR1;2 but not with the analogous domain of the SO₄²⁻ transporter isoform SULTR1;1, even though the SULTR1;1 STAS peptide also interacts with OASTL based on the yeast two-hybrid system and in vitro binding assays. These observations suggest a regulatory model in which interactions between SULTR1;2 and OASTL coordinate internalization of SO₄²⁻ with the energetic/metabolic state of plant root cells.     

Nitrogen Fixation Associated with the New Zealand Mangrove (Avicennia marina (Forsk.) Vierh. var. resinifera (Forst. f.) Bakh.)

Nitrogenase activity in mangrove forests at two locations in the North Island, New Zealand, was measured by acetylene reduction and ¹⁵N₂ uptake. Nitrogenase activity (C₂H₂ reduction) in surface sediments 0 to 10 mm deep was highly correlated (r = 0.91, n = 17) with the dry weight of decomposing particulate organic matter in the sediment and was independent of light. The activity was not correlated with the dry weight of roots in the top 10 mm of sediment (r = −0.01, n = 13). Seasonal and sample variation in acetylene reduction rates ranged from 0.4 to 50.0 μmol of C₂H₄ m⁻² h⁻¹ under air, and acetylene reduction was depressed in anaerobic atmospheres. Nitrogen fixation rates of decomposing leaves from the surface measured by ¹⁵N₂ uptake ranged from 5.1 to 7.8 nmol of N₂ g (dry weight)⁻¹ h⁻¹, and the mean molar ratio of acetylene reduced to nitrogen fixed was 4.5:1. Anaerobic conditions depressed the nitrogenase activity in decomposing leaves, which was independent of light. Nitrogenase activity was also found to be associated with pneumatophores. This activity was light dependent and was probably attributable to one or more species of Calothrix present as an epiphyte. Rates of activity were generally between 100 and 500 nmol of C₂H₄ pneumatophore⁻¹ h⁻¹ in summer, but values up to 1,500 nmol of C₂H₄ pneumatophore⁻¹ h⁻¹ were obtained.     

Estimation of Bacterial Nitrate Reduction Rates at In Situ Concentrations in Freshwater Sediments

A method was developed to follow bacterial nitrate reduction in freshwater sediments by using common high-performance liquid chromatographic equipment. The low detection limit (14 pmol) of the method enabled us to study concentration profiles and reaction kinetics under natural conditions. Significant nitrate concentrations (1 to 27 μM) were observed in the sediment of Lake Vechten during the nonstratified period; the concentration profiles showed a successive depletion of oxygen, nitrate, and sulfate with depth. The profiles were restricted to the upper 3 cm of the sediment which is rich in organics and loosely structured. Nitrate reduction in the sediment-water interface followed first-order reaction kinetics at in situ concentrations. Remarkably high potential nitrate-reducing activity was observed in the part of the sediment in which nitrate did not diffuse. This activity was also observed throughout the whole year. Estimates of K_m varied between 17 and 100 μM and V_max varied between 7.2 and 36 μmol cm⁻³ day⁻¹ for samples taken at different depths. The diffusion coefficient of nitrate ([10 ± 0.4] × 10⁻⁶ cm² s⁻¹) across the sediment-water interface was estimated by a constant-source technique and applied to a mathematical model to estimate the net nitrate reduction during the nonstratified period. In this period, observed nitrate reduction rates by the model, 0.2 to 0.4 mmol m⁻² day⁻¹, were lower than those found for oxygen (27 mmol m⁻² day⁻¹) and sulfate (0.4 mmol m⁻² day⁻¹). During the summer stratification, nitrate was absent in the sediment and reduction could not be estimated by the model.     

Biphasic Behavior of Anammox Regulated by Nitrite and Nitrate in an Estuarine Sediment

The production of N₂ gas via anammox was investigated in sediment slurries at in situ NO₂⁻ concentrations in the presence and absence of NO₃⁻. With single enrichment above 10 μM ¹⁴NO₂⁻ or ¹⁴NO₃⁻ and ¹⁵NH₄⁺, anammox activity was always linear (P < 0.05), in agreement with previous findings. In contrast, anammox exhibited a range of activity below 10 μM NO₂⁻ or NO₃⁻, including an elevated response at lower concentrations. With 100 μM NO₃⁻, no significant transient accumulation of NO₂⁻ could be measured, and the starting concentration of NO₂⁻ could therefore be regulated. With dual enrichment (1 to 20 μM NO₂⁻ plus 100 μM NO₃⁻), there was a pronounced nonlinear response in anammox activity. Maximal activity occurred between 2 and 5 μM NO₂⁻, but the amplitude of this peak varied across the study (November 2003 to June 2004). Anammox accounted for as much as 82% of the NO₂⁻ added at 1 μM in November 2003 but only for 15% in May 2004 and for 26 and 5% of the NO₂⁻ added at 5 μM for these two months, respectively. Decreasing the concentration of NO₃⁻ but holding NO₂⁻ at 5 μM decreased the significance of anammox as a sink for NO₂⁻. The behavior of anammox was explored by use of a simple anammox-denitrification model, and the concept of a biphasic system for anammox in estuarine sediments is proposed. Overall, anammox is likely to be regulated by the availability of NO₃⁻ and NO₂⁻ and the relative size or activity of the anammox population.     

Evidence for Autotrophy via the Reverse Tricarboxylic Acid Cycle in the Marine Magnetotactic Coccus Strain MC-1

Strain MC-1 is a marine, microaerophilic, magnetite-producing, magnetotactic coccus phylogenetically affiliated with the α-Proteobacteria. Strain MC-1 grew chemolithotrophically with sulfide and thiosulfate as electron donors with HCO₃⁻/CO₂ as the sole carbon source. Experiments with cells grown microaerobically in liquid with thiosulfate and H¹⁴CO₃⁻/¹⁴CO₂ showed that all cell carbon was derived from H¹⁴CO₃⁻/¹⁴CO₂ and therefore that MC-1 is capable of chemolithoautotrophy. Cell extracts did not exhibit ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO) activity, nor were RubisCO genes found in the draft genome of MC-1. Thus, unlike other chemolithoautotrophic, magnetotactic bacteria, strain MC-1 does not appear to utilize the Calvin-Benson-Bassham cycle for autotrophy. Cell extracts did not exhibit carbon monoxide dehydrogenase activity, indicating that the acetyl-coenzyme A pathway also does not function in strain MC-1. The ¹³C content of whole cells of MC-1 relative to the ¹³C content of the inorganic carbon source (Δδ¹³C) was −11.4 . Cellular fatty acids showed enrichment of ¹³C relative to whole cells. Strain MC-1 cell extracts showed activities for several key enzymes of the reverse (reductive) tricarboxylic acid (rTCA) cycle including fumarate reductase, pyruvate:acceptor oxidoreductase and 2-oxoglutarate:acceptor oxidoreductase. Although ATP citrate lyase (another key enzyme of the rTCA cycle) activity was not detected in strain MC-1 using commonly used assays, cell extracts did cleave citrate, and the reaction was dependent upon the presence of ATP and coenzyme A. Thus, we infer the presence of an ATP-dependent citrate-cleaving mechanism. These results are consistent with the operation of the rTCA cycle in MC-1. Strain MC-1 appears to be the first known representative of the α-Proteobacteria to use the rTCA cycle for autotrophy.     

Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the K_m was 39.9 mM, the V_max was 638 U mg⁻¹, and the K_cat/K_m was 1.79 × 10⁴ M⁻¹ s⁻¹, compared to a K_m of 76.0 mM, a V_max of 423 U mg⁻¹, and a K_cat/K_m of 0.62 × 10⁴ M⁻¹ s⁻¹ for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.     

Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

Mice deficient for the fibulin-5 gene (Fbln5^−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5^−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5^−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5^−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5^−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.     

Photoinhibition and P700 in the Marine Diatom Amphora sp

The marine diatom Amphora sp. was grown at a light intensity of 7.0 × 10¹⁵ quanta centimeter⁻² second⁻¹. Light saturation of photosynthesis for these cells was between 6.0 and 7.0 × 10¹⁶ quanta centimeter⁻² second⁻¹. At light intensities greater than saturation, photosynthetic ¹⁴CO₂ fixation was depressed, while P700 unit size (chlorophyll a concentration/P700 activity) increased and number of P700 units per cell decreased. After a 1-hour exposure of Amphora sp. to a photoinhibitory light intensity of 2.45 × 10¹⁷ quanta centimeter⁻² second⁻¹, there was a 45 to 50% decrease in the rate of ¹⁴CO₂ fixation relative to the rate at the culture light intensity. There also was a 25% increase in P700 unit size and a 30% reduction in the number of P700 units per cell but no change in total chlorophyll a concentration. Following this period of photoinhibition, the cells were returned to a light regime similar to that in the original culture conditions. Within 1 hour, both number of P700 units per cell and P700 unit size returned to levels similar to those of cells which were kept at the culture light intensity. The rates of photosynthesis did not recover as rapidly, requiring 2 to 3 hours to return to the rate for the nonphotoinhibited cells. Our results indicate that a decrease in P700 activity (with a resultant increase in P700 unit size) may be partially responsible for the photoinhibition of algal photosynthetic carbon dioxide fixation.     

Anion-Sensitive, H-Pumping ATPase of Oat Roots : Direct Effects of Cl, NO(3), and a Disulfonic Stilbene

To understand the mechanism and molecular properties of the tonoplast-type H⁺-translocating ATPase, we have studied the effect of Cl⁻, NO₃⁻, and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) on the activity of the electrogenic H⁺-ATPase associated with low-density microsomal vesicles from oat roots (Avena sativa cv Lang). The H⁺-pumping ATPase generates a membrane potential (Δψ) and a pH gradient (ΔpH) that make up two interconvertible components of the proton electrochemical gradient (μh⁺). A permeant anion (e.g. Cl⁻), unlike an impermeant anion (e.g. iminodiacetate), dissipated the membrane potential ([¹⁴C]thiocyanate distribution) and stimulated formation of a pH gradient ([¹⁴C]methylamine distribution). However, Cl⁻-stimulated ATPase activity was about 75% caused by a direct stimulation of the ATPase by Cl⁻ independent of the proton electrochemical gradient. Unlike the plasma membrane H⁺-ATPase, the Cl⁻-stimulated ATPase was inhibited by NO₃⁻ (a permeant anion) and by DIDS. In the absence of Cl⁻, NO₃⁻ decreased membrane potential formation and did not stimulate pH gradient formation. The inhibition by NO₃⁻ of Cl⁻-stimulated pH gradient formation and Cl⁻-stimulated ATPase activity was noncompetitive. In the absence of Cl⁻, DIDS inhibited the basal Mg,ATPase activity and membrane potential formation. DIDS also inhibited the Cl⁻-stimulated ATPase activity and pH gradient formation. Direct inhibition of the electrogenic H⁺-ATPase by NO₃⁻ or DIDS suggest that the vanadate-insensitive H⁺-pumping ATPase has anion-sensitive site(s) that regulate the catalytic and vectorial activity. Whether the anion-sensitive H⁺-ATPase has channels that conduct anions is yet to be established.     

Modifying Effect of a Common Polymorphism in the Interleukin-6 Promoter on the Relationship between Long-Term Exposure to Traffic-Related Particulate Matter and Heart Rate Variability

Background

Exposure to particulate matter (PM) has been associated with an increase in many inflammatory markers, including interleukin 6 (IL6). Air pollution exposure has also been suggested to induce an imbalance in the autonomic nervous system (ANS), such as a decrease in heart rate variability (HRV). In this study we aimed to investigate the modifying effect of polymorphisms in a major proinflammatory marker gene, interleukin 6 (IL6), on the relationship between long-term exposure to traffic-related PM₁₀ (TPM₁₀) and HRV.

Methods

For this cross-sectional study we analysed 1552 participants of the SAPALDIA cohort aged 50 years and older. Included were persons with valid genotype data, who underwent ambulatory 24-hr electrocardiogram monitoring, and reported on medical history and lifestyle. Main effects of annual average TPM₁₀ and IL6 gene variants (rs1800795; rs2069827; rs2069840; rs10242595) on HRV indices and their interaction with average annual exposure to TPM₁₀ were tested, applying a multivariable mixed linear model.

Results

No overall association of TPM₁₀ on HRV was found. Carriers of two proinflammatory G-alleles of the functional IL6 -174 G/C (rs1800795) polymorphism exhibited lower HRV. An inverse association between a 1 µg/m³ increment in yearly averaged TPM₁₀ and HRV was restricted to GG genotypes at this locus with a standard deviation of normal-to-normal intervals (SDNN) (GG-carriers: −1.8%; 95% confidence interval −3.5 to 0.01; p_{interaction(additive)} = 0.028); and low frequency power (LF) (GG-carriers: −5.7%; 95%CI: −10.4 to −0.8; p_{interaction(dominant)} = 0.049).

Conclusions

Our results are consistent with the hypothesis that traffic-related air pollution decreases heart rate variability through inflammatory mechanisms.     

Bacterium-Based NO2− Biosensor for Environmental Applications

A sensitive NO₂⁻ biosensor that is based on bacterial reduction of NO₂⁻ to N₂O and subsequent detection of the N₂O by a built-in electrochemical N₂O sensor was developed. Four different denitrifying organisms lacking NO₃⁻ reductase activity were assessed for use in the biosensor. The relevant physiological aspects examined included denitrifying characteristics, growth rate, NO₂⁻ tolerance, and temperature and salinity effects on the growth rate. Two organisms were successfully used in the biosensor. The preferred organism was Stenotrophomonas nitritireducens, which is an organism with a denitrifying pathway deficient in both NO₃⁻ and N₂O reductases. Alternatively Alcaligenes faecalis could be used when acetylene was added to inhibit its N₂O reductase. The macroscale biosensors constructed exhibited a linear NO₂⁻ response at concentrations up to 1 to 2 mM. The detection limit was around 1 μM NO₂⁻, and the 90% response time was 0.5 to 3 min. The sensor signal was specific for NO₂⁻, and interference was observed only with NH₂OH, NO, N₂O, and H₂S. The sensor signal was affected by changes in temperature and salinity, and calibration had to be performed in a system with a temperature and an ionic strength comparable to those of the medium analyzed. A broad range of water bodies could be analyzed with the biosensor, including freshwater systems, marine systems, and oxic-anoxic wastewaters. The NO₂⁻ biosensor was successfully used for long-term online monitoring in wastewater. Microscale versions of the NO₂⁻ biosensor were constructed and used to measure NO₂⁻ profiles in marine sediment.     

Solubilization and Characterization of a Membrane-Bound Auxin-Binding Protein from Cell Suspension Cultures of Nicotiana tabacum

A membrane-bound auxin-binding protein (MABP) was solubilized by Triton X-100 from cell suspension cultures of Nicotiana tabacum L. Solubilization of MABP was dependent on the detergent concentration and more than 80% of naphthalene-1-acetic acid (NAA)-binding activity was recovered by an optimum concentration of 0.2%. The solubilized MABP was highly heat-unstable and sensitive to protease. The properties of MABP (affinity, temperature dependence, pH optimum, and analog specificity for auxin binding) did not significantly change after solubilization, e.g. the solubilized MABP showed no or very low levels of NAA-binding at 0 to 4°C but showed a high-affinity binding (dissociation constant K_d = 2.7 ± 0.3 × 10⁻⁷m) at 25°C at an optimum pH of 5.0. NAA-binding of the solubilized MABP proceeded very slowly, i.e. a time of half-maximum binding was at least 15 minutes, although the solubilized MABP showed higher rates of association (k₁ = 1.3 versus 0.9 × 10⁵m⁻¹ min⁻¹) and dissociation (k₋₁ = 2.2 versus 1.6 × 10⁻² min⁻¹) with NAA than the bound MABP. These results show that specific, saturable, and reversible auxin binding to MABP from dicotyledonous N. tabacum differs from that from monocotyledonous Zea mays, and confirm that MABP is distinct from a soluble auxin-binding protein which also is present in N. tabacum.     

Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.