Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD(+)-dependent formate dehydrogenase

The NAD⁺-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

Effect of different levels of NADH availability on metabolite distribution in <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation in minimal and complex media

A range of intracellular NADH availability was achieved by combining external and genetic strategies. The effect of these manipulations on the distribution of metabolites in Escherichia coli was assessed in minimal and complex medium under anoxic conditions. Our in vivo system to increase intracellular NADH availability expressed a heterologous NAD⁺-dependent formate dehydrogenase (FDH) from Candida boidinii in E. coli. The heterologous FDH pathway converted 1 mol formate into 1 mol NADH and carbon dioxide, in contrast to the native FDH where cofactor involvement was not present. Previously, we found that this NADH regeneration system doubled the maximum yield of NADH from 2 mol to 4 mol NADH/mol glucose consumed. In the current study, we found that yields of greater than 4 mol NADH were achieved when carbon sources more reduced than glucose were combined with our in vivo NADH regeneration system. This paper demonstrates experimentally that different levels of NADH availability can be achieved by combining the strategies of feeding the cells with carbon sources which have different oxidation states and regenerating NADH through the heterologous FDH pathway. The general trend of the data is substantially similar for minimal and complex media. The NADH availability obtained positively correlates with the proportion of reduced by-products in the final culture. The maximum theoretical yield for ethanol is obtained from glucose and sorbitol in strains overexpressing the heterologous FDH pathway.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Ca<Superscript>2+</Superscript> and CaM are Involved in NO- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Adventitious Root Development in Marigold

Our previous results have demonstrated that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) are involved in the promotion of adventitious root development in marigold (Tagetes erecta L.). However, not much is known about the intricate molecular network of adventitious root development triggered by NO and H₂O₂. In this study, the involvement of calcium (Ca²⁺) and calmodulin (CaM) in NO- and H₂O₂-induced adventitious rooting in marigold was investigated. Exogenous Ca²⁺ was capable of promoting adventitious rooting, with a maximal biological response at 50 μM CaCl₂. Ca²⁺ chelators and CaM antagonists prevented NO- and H₂O₂-induced adventitious rooting, indicating that both endogenous Ca²⁺ and CaM may play crucial roles in the adventitious rooting induced by NO and H₂O₂. NO and H₂O₂ treatments increased the endogenous content of Ca²⁺ and CaM, suggesting that NO and H₂O₂ enhanced adventitious rooting by stimulating the endogenous Ca²⁺ and CaM levels. Moreover, treatment with Ca²⁺ enhanced the endogenous levels of NO and H₂O₂. Additionally, Ca²⁺ might be involved as an upstream signaling molecule for CaM during NO- and H₂O₂-induced rooting. Altogether, the results suggest that both Ca²⁺ and CaM are two downstream signaling molecules in adventitious rooting induced by NO and H₂O₂.     

Cloning and Characterization of a Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> Antiporter from Halophyte <Emphasis Type="Italic">Suaeda salsa</Emphasis> L.

We have isolated the cDNA of Ca²⁺/H⁺ antiporter which was designated as Suaeda salsa cation exchanger 1 (SsCAX1) from the C₃ halophyte S. salsa L. To ascertain the location of SsCAX1 in the cell, a peptide-specific antibody to SsCAX1 was prepared, and Western blotting analysis showed that it reacted only with a 48.8-kDa protein from S. salsa vacuolar membrane. Furthermore, SsCAX1 could resume yeast vacuolar Ca²⁺ transport mutants growth in high Ca²⁺ concentration (200 mM) culture medium. Northern blotting analysis showed that SsCAX1 expression was mainly found in the leaves and stems and slightly in the roots of S. salsa seedlings. Moreover, SsCAX1 expression levels and the protein amounts were significantly upregulated by CaCl₂ and NaCl treatment, respectively. In addition, the upregulation of the expression levels of V-H⁺-ATPase subunit c coordinated with the expression levels of the Ca²⁺/H⁺ antiporter under salinity. These results suggested that SsCAX1 from halophyte S. salsa might be a Ca²⁺ transporter at tonoplast and plays a key role in maintaining cytosolic Ca²⁺ homeostasis under saline condition.     

Transport of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> by an antiporter-related subunit from the <Emphasis Type="Italic">Escherichia coli </Emphasis>NADH dehydrogenase I produced in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na⁺ rather than H⁺. To elucidate the mechanism of Na⁺ transport, the C-terminally truncated NuoL subunit (NuoL_N) which is related to Na⁺/H⁺ antiporters was expressed as a protein A fusion protein (ProtA–NuoL_N) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoL_N catalyzed the uptake of Na⁺ and K⁺ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoL_N translocated Na⁺ and K⁺ independently from other complex I subunits. Na⁺ transport by ProtA–NuoL_N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na⁺/H⁺ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript> production within tumor microenvironment inversely correlated with infiltration of CD56<Superscript>dim</Superscript> NK cells in gastric and esophageal cancer: possible mechanisms of NK cell dysfunction

Human NK cells can be divided into two subsets, CD56^dimCD16(+)NK and CD56^brightCD16(−)NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed the relationship between CD56^dim/CD56^bright NK cells and H₂O₂ in tumor-infiltrating NK cells in patients with gastric (n = 50) and esophageal (n = 35) cancer. The ratio of CD56^dim NK cells infiltrating tumors gradually decreased according to disease progression. H₂O₂ was abundantly produced within tumor microenvironments, and there was an inverse correlation between CD56^dim NK cell infiltration and H₂O₂ production. CD56^dim NK cells are more sensitive to apoptosis induced by physiological levels of H₂O₂ than CD56^bright NK cells. Furthermore, the exposure of NK cells to H₂O₂ resulted in the impairment of ADCC activity. In conclusion, H₂O₂ produced within tumor microenvironments inversely correlated with the infiltration of CD56^dim NK cells, possibly due to their preferentially induced cell death. These observations may explain one of the mechanisms behind NK cell dysfunction frequently observed in tumor microenvironments.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Comparison of <Superscript>13</Superscript>CH<Subscript>3</Subscript>, <Superscript>13</Superscript>CH<Subscript>2</Subscript>D,and <Superscript>13</Superscript>CHD<Subscript>2</Subscript> methyl labeling strategies in proteins

A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins, using ¹³CH₃,¹³CH₂D, and ¹³CHD₂ methyl isotopomers, is presented. For each labeling scheme, ¹H–¹³C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue enzyme, malate synthase G, with ¹³CH₃,¹³CH₂D, and ¹³CHD₂ labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence has previously been identified as a transverse relaxation optimized experiment for ¹³CH₃-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (¹³CH₃ HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version of the ¹³CH₃ HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both ¹³C and ¹H spins. The HSQC and HMQC spectra of ¹³CH₂D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, ¹³CH₂D TROSY, that exploits cross-correlation effects to record ¹H–¹³C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of ¹³CHD₂-labeled methyl groups can be recorded with HSQC or HMQC; a new ¹³CHD₂ HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here. When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the ¹³CH₃ labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either ¹³CH₂D or ¹³CHD₂ labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented here.     

Resolution-optimized NMR measurement of <Superscript>1</Superscript>D<Subscript>CH</Subscript>, <Superscript>1</Superscript>D<Subscript>CC</Subscript> and <Superscript>2</Superscript>D<Subscript>CH</Subscript> residual dipolar couplings in nucleic acid bases

New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond ²D_CH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in ¹³C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear ¹H-¹H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven ¹H-¹³C and ¹³C-¹³C couplings are measured for pyrimidines (U and C), including ¹D_C5H5, ¹D_C6H6, ²D_C5H6, ²D_C6H5, ¹D_C5C4, ¹D_C5C6, and ²D_C4H5. For adenine, four base couplings (¹D_C2H2, ¹D_C8H8, ¹D_C4C5, and ¹D_C5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (¹D_C8H8, ¹D_C4C5, and ¹D_C5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide.     

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.     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Structure determination of proteins in <Superscript>2</Superscript>H<Subscript>2</Subscript>O solution aided by a deuterium-decoupled 3D HCA(N)CO experiment

We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone ¹Hα and ¹³Cα to those of ¹³C′ in the preceding residue. By applying ²H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO, HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone ¹³C′ and amide ¹⁵N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for ¹³C/¹⁵N-labeled GB1 dissolved in ²H₂O. The quality of the GB1 structure determined in ²H₂O was similar to that determined in H₂O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures in ²H₂O or H₂O at high pH values.     

Programmed cell death in plants: Protective effect of phenolic compounds against chitosan and H<Subscript>2</Subscript>O<Subscript>2</Subscript>

Addition of chitosan or H₂O₂ caused destruction of nuclei of epidermal cells (EC) in the epidermis isolated from pea leaves. Phenol, a substrate of the apoplastic peroxidase-oxidase, in concentrations of 10⁻¹⁰–10⁻⁶ M prevented the destructive effect of chitosan. Phenolic compounds 2,4-dichlorophenol, catechol, and salicylic acid, phenolic uncouplers of oxidative phosphorylation pentachlorophenol and 2,4-dinitrophenol, and a non-phenolic uncoupler carbonyl cyanide m-chlorophenylhydrazone, but not tyrosine or guaiacol, displayed similar protective effects. A further increase in concentrations of the phenolic compounds abolished their protective effects against chitosan. Malate, a substrate of the apoplastic malate dehydrogenase, replenished the pool of apoplastic NADH that is a substrate of peroxidase-oxidase, prevented the chitosan-induced destruction of the EC nuclei, and removed the deleterious effect of the increased concentration of phenol (0.1 mM). Methylene Blue, benzoquinone, and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) capable of supporting the optimal catalytic action of peroxidase-oxidase cancelled the destructive effect of chitosan on the EC nuclei. The NADH-oxidizing combination of TMPD with ferricyanide promoted the chitosan-induced destruction of the nuclei. The data suggest that the apoplastic peroxidase-oxidase is involved in the antioxidant protection of EC against chitosan and H₂O₂.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Differential Effects of Methionine and Cysteine Oxidation on [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Cultured Hippocampal Neurons

Methionine and cysteine residues in proteins are the major targets of reactive oxygen species (ROS). The present work was designed to characterize the impact of methionine and cysteine oxidation upon [Ca²⁺]_i in hippocampal neurons. We investigated the effects of H₂O₂ and chloramine T(Ch-T) agents known to oxidize both cysteine and methionine residues, and 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB)—a cysteine-specific oxidant, on the intracellular calcium in hippocampal neurons. The results showed that these three oxidants, 1 mM H₂O₂, 1 mM Ch-T, and 500 μM DTNB, induced an sustained elevation of [Ca²⁺]_i by 76.1 ± 3.9%, 86.5 ± 5.0%, and 24.4 ± 3.2% over the basal level, respectively. The elevation induced by H₂O₂ and Ch-T was significantly higher than DTNB. Pretreatment with reductant DTT at 1 mM for 10 min completely prevented the action of DTNB on [Ca²⁺]_i, but only partially reduced the effects of H₂O₂ and Ch-T on [Ca²⁺]_i, the reductions were 44.6 ± 4.2% and 29.6 ± 6.1% over baseline, respectively. The elevation of [Ca²⁺]_i induced by H₂O₂ and Ch-T after pretreatment with DTT were statistically higher than that induced by single administration of DTNB. Further investigation showed that the elevation of [Ca²⁺]_i mainly resulted from internal calcium stores. From our data, we propose that methionine oxidation plays an important role in the regulation of intracellular calcium and this regulation may mainly be due to internal calcium stores.     

The interaction between H<Subscript>2</Subscript>O<Subscript>2</Subscript> and NO,Ca<Superscript>2+</Superscript>, cGMP,and MAPKs during adventitious rooting in mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In the present study, we present a signaling network involving H₂O₂, nitric oxide (NO), calcium (Ca²⁺), cyclic guanosine monophosphate (cGMP), and the mitogen-activated protein kinase (MAPK) cascade during adventitious rooting in mung bean seedlings. Both exogenous H₂O₂ and the NO donor sodium nitroprussiate were capable of promoting the formation and development of adventitious roots. H₂O₂ and NO signaling pathways were elicited in parallel in auxin-induced adventitious rooting. Cytosolic Ca²⁺ was required for adventitious rooting, and Ca²⁺ served as a downstream component of H₂O₂, as well as cGMP or MAPK, signaling cascades. cGMP and MAPK cascades function downstream of H₂O₂ signaling and depend on auxin responses in adventitious root signaling processes.