Inhibition of hydrogen uptake in <Emphasis Type="Italic">Escherichia coli</Emphasis> by expressing the hydrogenase from the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis>sp. PCC 6803

Background  

Molecular hydrogen is an environmentally-clean fuel and the reversible (bi-directional) hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 as well as the native Escherichia coli hydrogenase 3 hold great promise for hydrogen generation. These enzymes perform the simple reaction 2H⁺ + 2e^- ↔ H₂ (g).     

Identification of a new 130 bp <Emphasis Type="Italic">cis</Emphasis>-acting element in the <Emphasis Type="Italic">TsVP1</Emphasis> promoter involved in the salt stress response from <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Background  

Salt stress is one of the major abiotic stresses affecting plant growth and productivity. Vacuolar H⁺-pyrophosphatase (H⁺-PPase) genes play an important role in salt stress tolerance in multiple species.     

Divergent calcium signaling in RBCs from <Emphasis Type="Italic">Tropidurus torquatus</Emphasis> (Squamata – Tropiduridae) strengthen classification in lizard evolution

Background  

We have previously reported that a Teiid lizard red blood cells (RBCs) such as Ameiva ameiva and Tupinambis merianae controls intracellular calcium levels by displaying multiple mechanisms. In these cells, calcium stores could be discharged not only by: thapsigargin, but also by the Na⁺/H⁺ ionophore monensin, K⁺/H⁺ ionophore nigericin and the H⁺ pump inhibitor bafilomycin as well as ionomycin. Moreover, these lizards possess a P2Y-type purinoceptors that mobilize Ca²⁺ from intracellular stores upon ATP addition.     

The Vacuolar ATPase from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>: Molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein

Background  

Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H⁺-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes.     

Growth and physiological activity in <Emphasis Type="Italic">Larix kaempferi</Emphasis> seedlings inoculated with ectomycorrhizae as affected by soil acidification

To evaluate the effect of ectomycorrhizal colonization on growth and physiological activity of Larix kaempferi seedlings grown under soil acidification, we grew L. kaempferi seedlings with three types of ectomycorrhizae for 180 days in acidified brown forest soil derived from granite. The soil had been treated with an acid solution (0 (control), 10, 30, 60, and 90 mmol H⁺ kg⁻¹). The water-soluble concentrations of Ca, Mg, K, Al, and Mn increased with increasing amounts of H⁺ added to the soil. Ectomycorrhizal development significantly increased in soil treated with 10 and 30 mmol H⁺ kg⁻¹ but was significantly reduced in soil treated with 60 and 90 mmol H⁺ kg⁻¹. The concentrations of Al and Mn in needles or roots increased with increasing H⁺ added to the soil. The total N in seedlings significantly increased with increasing H⁺ in soil and colonization with ectomycorrhiza. The maximum net photosynthetic rate at light and CO₂ saturation (P _max) was greater in soil treated with 10 mmol H⁺ kg⁻¹ than in controls, and was less is soils treated with greater than with 30 mmol H⁺ kg⁻¹, especially with 60 and 90 mmol H⁺ kg⁻¹. However, colonization with ectomycorrhiza significantly reduced the concentration of Al and Mn in needles or roots and increased the values of P _max and total dry mass (TDM). The relative TDM of L. kaempferi seedlings was approximately 40% at a (BC, base cation)/Al ratio of 1.0. However, ectomycorrhizal seedlings had a 100–120% greater TDM at a BC/Al ratio of 1.0 than non-ectomycorrhizal seedlings, even though the acid treatment reduced their overall growth.     

Ethylene and nitric oxide are involved in maintaining ion homeostasis in <Emphasis Type="Italic">Arabidopsis</Emphasis> callus under salt stress

In the present study, the role of ethylene in nitric oxide (NO)-mediated protection by modulating ion homeostasis in Arabidopsis callus under salt stress was investigated. Results showed that the ethylene-insensitive mutant etr1-3 was more sensitive to salt stress than the wild type (WT). Under 100 mM NaCl, etr1-3 callus displayed a greater electrolyte leakage and Na⁺/K⁺ ratio but a lower plasma membrane (PM) H⁺-ATPase activity compared to WT callus. Application of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor) or sodium nitroprusside (SNP, a NO donor) alleviated NaCl-induced injury by maintaining a lower Na⁺/K⁺ ratio and an increased PM H⁺-ATPase activity in WT callus but not in etr1-3 callus. The SNP actions in NaCl stress were attenuated by a specific NO scavenger or an ethylene biosynthesis inhibitor in WT callus. Under 100 mM NaCl, the NO accumulation and ethylene emission appeared at early time, and NO production greatly stimulated ethylene emission in WT callus. In addition, ethylene induced the expression of PM H⁺-ATPase genes under salt stress. The recovery experiment showed that NaCl-induced injury was reversible, as signaled by the similar recovery of Na⁺/K⁺ ratio and PM H⁺-ATPase activity in WT callus. Taken together, the results indicate that ethylene and NO cooperate in stimulating PM H⁺-ATPase activity to modulate ion homeostasis for salt tolerance, and ethylene may be a part of the downstream signal molecular in NO action.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.     

Metagenome Cloning and Functional Analysis of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Genes from Keke Salt Lake in China

Na⁺/H⁺ antiporters are ubiquitous membrane proteins and play a central role in cell homeostasis including pH regulation, osmoregulation, and Na⁺/Li⁺ tolerance in bacteria. The microbial communities in extremely hypersaline soil are an important resource for isolating Na⁺/H⁺ antiporter genes. A metagenomic library containing 35,700 clones was constructed by using genomic DNA obtained from the hypersaline soil samples of Keke Salt Lake in Northwest of China. Two Na⁺/H⁺ antiporters, K1-NhaD, and K2-NhaD belonging to NhaD family, were screened and cloned from this metagenome by complementing the triple mutant Escherichia coli strain KNabc (nhaA ⁻ , nhaB ⁻ , chaA ⁻) in medium containing 0.2 M NaCl. K1-NhaD and K2-NhaD have 75.5% identity at the predicted amino acid sequence. K1-NhaD has 78% identity with Na⁺/H⁺ antiporter NhaD from Halomonas elongate at the predicted amino acid sequence. The predicted K1-NhaD is a 53.5 kDa protein (487 amino acids) with 13 transmembrane helices. K2-NhaD has 73% identity with Alkalimonas amylolytica NhaD. The predicted K2-NhaD is a 55 kDa protein (495 amino acids) with 12 transmembrane helices. Both K1-NhaD and K2-NhaD could make the triple mutant E. coli KNabc (nhaA ⁻ , nhaB ⁻ , chaA⁻) grow in the LBK medium containing 0.2–0.6 M Na⁺ or with 0.05–0.4 M Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying K1-NhaD or K2-NhaD exhibited Na⁺/H⁺ and Li⁺/H⁺ antiporter activities which were pH-dependent with the highest activity at pH 9.5. Little K⁺/H⁺ antiporter activity was also detected in vesicles form E. coli KNabc carrying K1-NhaD or K2-NhaD.     

Temperature limited fed-batch technique for control of proteolysis in <Emphasis Type="Italic">Pichia pastoris</Emphasis> bioreactor cultures

Background  

A temperature limited fed-batch (TLFB) technique is described and used for Pichia pastoris Mut⁺ strain cultures and compared with the traditional methanol limited fed-batch (MLFB) technique. A recombinant fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A and lipase B from Candida antarctica (CALB), was produced and secreted by this strain.     

Real‐time PCR for Helicobacter pylori diagnosis. The best tools available

Background

Knowledge of antimicrobial susceptibility, especially to macrolides, has become crucial for the management of Helicobacter pylori infection. Our aim was to evaluate two new PCR kits able to detect H. pylori in gastric biopsies as well as the mutations associated with macrolide resistance.

Materials and Methods

Two hundred successive biopsies (received from gastroenterologists all over France) were used. The two new kits tested were Amplidiag H. pylori+Clari^R from Mobidiag Espoo, Finland, and RIDA^®GENE H. pylori from R‐Biopharm, Darmstadt, Germany. Culture and a validated in‐house real‐time PCR were also performed, and in the case of a positive culture, Etest for clarithromycin was carried out. Discrepancies were solved by looking at the pathologic data.

Results

Culture was positive in 68 cases (34%), and with our in‐house real‐time PCR in these 68 cases plus 5 others (N = 73, 36%). All were also detected by the two new kits. In addition, RIDA^®GENE H. pylori detected one more positive also detected by Amplidiag H. pylori+Clari^R, and Amplidiag detected two other positives. Of these three additional cases, pathology confirmed the positivity for two. Only one case diagnosed by Amplidiag could be considered as a false positive. With regard to clarithromycin resistance, 22 cases were detected. The corresponding mutations (A2142/43G) were all identified with the three PCRs.

Conclusions

These two new kits which have an excellent sensitivity and specificity are convenient to use, adaptable to different thermocyclers, provide quick results, and deserve to be used in H. pylori diagnosis for a better choice of treatment regimen.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

Eu3+‐tetrakis β‐diketonate complexes for solid‐state lighting application

Eu³⁺–β‐diketonate complexes are used, for example, in solid‐state lighting (SSL) or light‐converting molecular devices. However, their low emission quantum efficiency due to water molecules coordinated to Eu³⁺ and low photostability are still problems to be addressed. To overcome such challenges, we synthesized Eu³⁺ tetrakis complexes based on [Q][Eu(tfaa)₄] and [Q][Eu(dbm)₄] (Q1 = C₂₆H₅₆N⁺, Q2 = C₁₉H₄₂N⁺, and Q3 = C₁₇H₃₈N⁺), replacing the water molecules in the tris stoichiometry. The tetrakis β‐diketonates showed desirable thermal stability for SSL and, under excitation at 390 nm, they displayed the characteristic Eu³⁺ emission in the red spectral region. The quantum efficiencies of the dbm complexes achieved values as high as 51%, while the tfaa complexes exhibited lower quantum efficiencies (28–33%), but which were superior to those reported for the tris complexes. The structures were evaluated using the Sparkle/PM7 model and comparing the theoretical and the experimental Judd–Ofelt parameters. [Q1][Eu(dbm)₄] was used to coat a near‐UV light‐emitting diode (LED), producing a red‐emitting LED prototype that featured the characteristic emission spectrum of [Q1][Eu(dbm)₄]. The emission intensity of this prototype decreased only 7% after 30 h, confirming its high photostability, which is a notable result considering Eu³⁺ complexes, making it a potential candidate for SSL.