Naphthalene/quinoline amides and sulfonylureas as potent and selective antagonists of the EP4 receptor

Two new series of EP₄ antagonists based on naphthalene/quinoline scaffolds have been identified as part of our on-going efforts to develop treatments for inflammatory pain. One series contains an acidic sulfonylurea pharmacophore, whereas the other is a neutral amide. Both series show subnanomolar intrinsic binding potency towards the EP₄ receptor, and excellent selectivity towards other prostanoid receptors. While the amide series generally displays poor pharmacokinetic parameters, the sulfonylureas exhibit greatly improved profile. MF-592, the optimal compound from the sulfonylurea series, has a desirable overall preclinical profile that suggests it is suitable for further development.     

Novel H3 receptor antagonists with improved pharmacokinetic profiles

A new series of H₃ antagonists derived from the natural product Conessine are presented. Several compounds from these new series retain the potency and selectivity of earlier diamine based analogs while exhibiting improved PK characteristics. One compound (3u) demonstrated functional antagonism of the H₃ receptor in an in vivo pharmacological model.     

The capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential of the terminal electron acceptor

The effect of different electron acceptors on substrate degradation was studied in pure and mixed cultures of various hydrogenotrophic homoacetogenic, methanogenic, sulfate-reducing, fumarate-reducing and nitrate-ammonifying bacteria. Two different species of these bacteria which during organic substrate degradation produce and consume hydrogen, were cocultured on a substrate which was utilized only by one of them. Hydrogen, which was excreted as intermediate by the first strain (and reoxidized in pure culture), could, depending on the hydrogen acceptor present, also be used by the second organism, resulting in interspecies hydrogen transfer. The efficiency of H₂ transfer was similar when methanol, lactate or fructose were used as organic substrate, although the free energy changes of fermentative H₂ formation of these substrates are considerably different. In coculture experiments nitrate or fumarate>sulfate> CO₂/CH₄>sulfur or CO₂/acetate were the preferred electron acceptors, and an increasing percentage of H₂ was transferred to that bacterium which was able to utilize the preferred electron acceptor. In pure culture the threshold values for hydrogen oxidation decreased in the same order from 1,100 ppm for homoacetogenic bacteria to about 0.03 ppm for nitrate or fumarate reducing bacteria. The determined H₂-threshold values as well as the percentage of H₂ transfer in cocultures were related to the Gibbs free energy change of the respective hydrogen oxidizing reaction.Parts of this work (grant to R C-R) was supported by the European Community (ST2A-0022)     

Origin and evolution of photosynthetic reaction centers

The prototype reaction center may have used protoporphyrin-IX associated with small peptides to transfer electrons or protons across the primitive cell membrane. The precursor of all contemporary reaction centers contained chlorophylla molecules as both primary electron donor and initial electron acceptor and an Fe-S center as secondary acceptor (RC-1 type). The biosynthetic pathway for chlorophylla evolved along with the evolution of a better organized reaction center associated with cytochromes and quinones in a primitive cyclic electron transport system. This reaction center probably functioned initially in photoassimilation, but was easily adapted to CO₂ fixation using H₂ and H₂S as reductants. During this phase bacteriochlorophyllg may have evolved from chlorophylla in response to competition for light, and thereby initiated the gram-positive line of eubacteria. A second reaction center (RC-2) evolved from RC-1 between 3.5 and 2.5 Ga ago in response to the competition for reductants for CO₂ fixation. The new organism containing RC-2 in series with RC-1 would have been able to use poor reducing agents such as the abundant aqueous ferrous ion in place of H₂ and H₂S. This new organism is proposed to be the common ancestor of all phototrophic eubacteria except those related to the gram-positive bacteria. All organisms containing bacteriochlorophylla lost either RC-1 or RC-2, while those organisms containing chlorophylla (ancestors of cyanobacteria) added a water-splitting enzyme to RC-2 between 3.0 and 2.5 Ga ago in order to use H₂O in place of hydrated ferrous ion as electron donor for autotrophic photosynthesis.     

Stimulating the anaerobic degradation of aromatic hydrocarbons in contaminated sediments by providing an electrode as the electron acceptor

The possibility that electrodes might serve as an electron acceptor to simulate the degradation of aromatic hydrocarbons in anaerobic contaminated sediments was investigated. Initial studies with Geobacter metallireducens demonstrated that although toluene was rapidly adsorbed onto the graphite electrodes it was rapidly oxidized to carbon dioxide with the electrode serving as the sole electron acceptor. Providing graphite electrodes as an electron acceptor in hydrocarbon‐contaminated sediments significantly stimulated the removal of added toluene and benzene. Rates of toluene and benzene removal accelerated with continued additions of toluene and benzene. [¹⁴C]‐Toluene and [¹⁴C]‐benzene were quantitatively recovered as [¹⁴C]‐CO₂, demonstrating that even though the graphite adsorbed toluene and benzene they were degraded. Introducing an electrode as an electron acceptor also accelerated the loss of added naphthalene and [¹⁴C]‐naphthalene was converted to [¹⁴C]‐CO₂. The results suggest that graphite electrodes can serve as an electron acceptor for the degradation of aromatic hydrocarbon contaminants in sediments, co‐localizing the contaminants, the degradative organisms and the electron acceptor. Once in position, they provide a permanent, low‐maintenance source of electron acceptor. Thus, graphite electrodes may offer an attractive alternative for enhancing contaminant degradation in anoxic environments.     

Identical Ring Cleavage Products during Anaerobic Degradation of Naphthalene, 2-Methylnaphthalene, and Tetralin Indicate a New Metabolic Pathway

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C₁ unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C₁₁H₁₆O₄ (C₁₁H₁₆O₄-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by β-oxidation of one of the side chains of the C₁₁H₁₆O₄-diacid. Stable isotope-labeling growth experiments with either ¹³C-labeled naphthalene, per-deuterated naphthalene-d₈, or a ¹³C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.     

Electron flow accompanying the ascorbate peroxidase cycle in maize mesophyll chloroplasts and its cooperation with linear electron flow to NADP+ and cyclic electron flow in thylakoid membrane energization

Potential roles for cyclic and pseudocyclic electron flow in C₄ plants are to provide ATP for the C₄ cycle and, under excess light, to down-regulate PS II activity through membrane energization. Intact mesophyll chloroplasts of maize were used to evaluate forms of electron transport including the Mehler peroxidase reaction (linear electron flow to O₂, formation of H₂O₂ which is reduced by ascorbate, and linear flow linked to reduction of oxidized ascorbate). Addition of H₂O₂ to isolated chloroplasts in the light in the presence of an uncoupler induced Photosystem (PS) II activity, as determined from increases in photochemical quenching of chlorophyll fluorescence (q_p) and the quantum yield of PS II. H₂O₂ also induced dissipation of energy by thylakoid membrane energization and non-photochemical fluorescence quenching (q_n), which was inhibited by addition of an uncoupler. These effects of H₂O₂ on q_p and q_n were inhibited by addition of KCN, an inhibitor of ascorbate peroxidase. The results suggest that H₂O₂ is reduced via ascorbate, and that the oxidized ascorbate is then reduced by linear electron flow contributing to photochemistry and thylakoid membrane energization. Evidence for function of pseudocyclic electron flow via the Mehler peroxidase reaction was obtained with only oxygen as an electron acceptor, as well as in the presence of oxaloacetate a natural electron acceptor in C₄ photosynthesis. KCN decreased q_p and PS II yield in the absence and presence of oxaloacetate and, in the former case, it severely reduced q_{_n}. KCN also decreased pH formation across the thylakoid membrane based on its decrease in the light-induced quenching of 9-aminoacridine fluorescence, particularly in the absence of oxaloacetate. Antimycin A, an inhibitor of cyclic electron flow, also diminished pH formation. These results provide evidence for shared energization of thylakoid membranes by the Mehler peroxidase reaction, cyclic electron flow, and linear electron flow linked to the C₄ pathway.     

Soil H2-uptake in relation to soil properties and rhizobial H2-production

Summary The biological nature of soil H₂-consumption has been investigated. Soil microorganisms were capable to remove H₂ present in the gas phase at concentrations in the range of 200 ppm at rates varying between 0.2 and 1.0 l.min^–1. 100 g^–1. Free soil enzymes did not contribute significantly at the H₂ concentrations tested. Oxygen seemed to be the predominant electron acceptor. The influence of microbiological and physical soil properties on the H₂-uptake activity was examined for 38 soils.A highly significant correlation between biomass-C and H₂-uptake rate of the soil was noted, suggesting that the latter parameter might be useful as an indirect estimation of soil microbial biomass. The correlation was however not applicable for soils recently grown with legumes. Indeed, soya plants nodulated with aRhizobium strain with a weak hydrogen uptake capability, strongly increased the hydrogen oxidizing capability of the surrounding soil.     

Purification and Characterization of Membrane-Associated Hydrogenase from the Deep-Sea Epsilonproteobacterium Hydrogenimonas thermophila

Membrane-associated hydrogenase was purified from the chemolithoautotrophic epsilonproteobacterium Hydrogenimonas thermophila at 152-fold purity. The hydrogenase was found to be localized in the periplasmic space, and was easily solubilized with 0.1% Triton X-100 treatment. Hydrogen oxidation activity was 1,365 μmol H₂/min/mg of protein at 80 °C at pH 9.0, with phenazine methosulphate as the electron acceptor. Hydrogen production activity was 900 μmol H₂/min/mg of protein at 80 °C and pH 6.0, with reduced methyl viologen as the electron donor. The hydrogenase from this organism showed higher oxygen tolerance than those from other microorganisms showing hydrogen oxidation activity. The structural genes of this hydrogenase, which contains N-terminal amino acid sequences from both small and large subunits of purified hydrogenase, were successfully elucidated. The hydrogenase from H. thermophila was found to be phylogenetically related with H₂ uptake hydrogenases from pathogenic Epsilonproteobacteria.     

Enhanced detection of H2O2 in cells expressing Horseradish Peroxidase

A new procedure for fluorescent detection of intracellular H₂O₂ in cells transiently expressing the catalyst Horseradish Peroxidase (HRP) is setup and validated. More specific reaction with HRP largely amplifies oxidation of the redox probes used (2′,7′-dichlorodihydrofluorescein and dihydrorhodamine). Expression of HRP does not affect cell viability. The procedure reveals MAO activity, a primary intracellular H₂O₂ source, in monolayers of intact transfected cells. The probes oxidation rate responds specifically to the MAO activation/inhibition. Their oxidation by MAO-derived H₂O₂ is sensitive to intracellular H₂O₂ competitors: it decreases when H₂O₂ is removed by pyruvate and it increases when the GSH-dependent removal systems are impaired. Specific response was also measured after addition of extracellular H₂O₂. Oxidation of the fluorescent probes following reaction of H₂O₂ with endogenous HRP overcomes most criticisms in their use for intracellular H₂O₂ detection. The method can be applied for direct determination in plate reader and is proposed to detect H₂O₂ generation in physio-pathological cell models.     

Behaviour of toluene, benzene and naphthalene under anaerobic conditions in sediment columns

The biotransformation of toluene, benzene and naphthalene was examined in anaerobic sediment columns. Five columns filled with a mixture of sediments were operated in the presence of bicarbonate, sulfate, iron, manganese, or nitrate as electron acceptor. The columns were continuously percolated with a mixture of the three organic compounds (individual concentrations 25–200 μM) at 20°C. Toluene was transformed readily (within 1 to 2 months) under all redox conditions tested. Benzene was recalcitrant over the test period of 375–525 days in all five columns. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect was observed after adding and omitting an easier degradable substrate. [¹⁴C]naphthalene was used to confirm this disappearance to be the result of degradation; two third of the naphthalene was converted to CO₂.     

Computational exploration of the gas adsorption on the iron tetracarboxylate metal-organic framework MIL-102

Density functional theory calculations have been combined with forcefield-based grand canonical Monte Carlo simulations to explore the adsorption of CO₂, N₂, CH₄ and H₂ on the small one-dimensional channel MIL-102, a naphthalene tetracarboxylate-based metal-organic framework (MOF) built up from a connection of trimers of trivalent iron. A detailed analysis is provided on the preferential arrangement of the confined adsorbates as well as the energetics of the host/guest interactions. The co-adsorption properties of this solid for the elimination of CO₂ from hydrogen, natural and flue gases are then revealed. The so-predicted performances are further compared with those reported so far for a diverse series of MOFs.     

Enhancement of BKCa channel activity induced by hydrogen peroxide: Involvement of lipid phosphatase activity of PTEN

Large-conductance calcium and voltage-dependent potassium (BK_Ca) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H₂O₂) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hβ1 were cotransfected with the PTEN_wt, PTEN_C124S, PTEN_G129E in HEK 293 cells. Typical BK_Ca channel currents could be recorded in cell-attached configurations. We found that PTEN_wt reduced the H₂O₂-induced BK_Ca channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN_C124S/PTEN_G129E mutants that lack lipid phosphatase activity produced no regulation on the H₂O₂-induced BK_Ca channel activation. These results demonstrated that PTEN regulated the H₂O₂-induced BK_Ca channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H₂O₂-induced BK_Ca channel activation was attenuated when cells were treated with H₂O₂ at concentrations higher than 100 μM or at 100 μM for long-term treatment. In addition, the p-AKT expression level in PTEN_wt overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca²⁺]_i) induced by H₂O₂ was also inhibited. These findings may elucidate a new mechanism for H₂O₂-induced BK_Ca channel activation and provide some evidences for the role of PTEN on vasodilation induced by H₂O₂.     

Behaviour of toluene, benzene and naphthalene under anaerobic conditions in sediment columns

The biotransformation of toluene, benzene and naphthalene was examined in anaerobic sediment columns. Five columns filled with a mixture of sediments were operated in the presence of bicarbonate, sulfate, iron, manganese, or nitrate as electron acceptor. The columns were continuously percolated with a mixture of the three organic compounds (individual concentrations 25–200 M) at 20°C.Toluene was transformed readily (within 1 to 2 months) under all redox conditions tested. Benzene was recalcitrant over the test period of 375–525 days in all five columns. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect was observed after adding and omitting an easier degradable substrate. [¹⁴C]naphthalene was used to confirm this disappearance to be the result of degradation; two third of the naphthalene was converted to CO₂.     

Isolation and characterization of hydrogenase-negative mutants of Azorhizobium caulinodans ORS571

Hydrogenase-negative (Hup^-) mutants of Azorhizobium caulinodans ORS571 were isolated by means of Tn5 mutagenesis. The colony test used for screening for Hup^- strains was based on the absence of reduction of triphenyltetrazolium chloride with hydrogen. Suspensions from cultures of the mutant strains grown under derepressing conditions did not use hydrogen with methylene blue or oxygen as the hydrogen acceptor. The mutants were shown to carry single Tn5 insertions at different locations in the A. caulinodans genome. Molar growth yields (corrected for poly--hydroxybutyrate formation) in chemostat cultures of the mutants were similar to those of the wild type. Molar growth yields of the mutants were not increased by passing additional hydrogen through chemostat cultures, which is in agreement with the hydrogenase-negative phenotype of the mutants. H₂/N₂ ratios (mol H₂ formed per mol N₂ fixed) were calculated from the hydrogen content of the effluent gas and the N-content of the bacterial dry weight. Low H₂/N₂ ratios (between 1.2 and 1.9) were found in both energy-limited (oxygen or succinate) cultures and in cultures limited by the supply of an anabolic substrate (Mg²⁺). ATP/2e values (mol ATP used at the transport of 2e to nitrogen or H⁺) were calculated from the H₂/N₂ ratios and the molar growth yields of nitrogen-fixing and ammonia-assimilating cultures. ATP/2e values were between 7 and 11. It was concluded that the calculated ATP/2e values comprise not only 4 mol ATP used at the transport of 2e through nitrogenase but also energy equivalents needed for reversed electron flow from NADH to the low-potential hydrogen donor used by nitrogenase.     

Study on the heterogeneous degradation of chitosan with H2O2 catalyzed by a new supermolecular assembly crystal: [C6H8N2]6H3[PW12O40]·2H2O

A new supermolecular assembly crystal, [C₆H₈N₂]₆H₃[PW₁₂O₄₀]·2H₂O (DMB-PWA), was synthesized with phosphotungstic acid (PWA) and 1,2-diaminobenzene (DMB) under hydrothermal conditions and was characterized by Fourier-transform infrared spectra (FTIR) and single-crystal X-ray diffraction analysis. DMB-PWA could effectively catalyze oxidative degradation of chitosan with H₂O₂ in the heterogeneous phase. The optimum degradation conditions were determined by orthogonal tests as follows: amount of chitosan 1.00 g, 30% (wt %); H₂O₂, 3.0 mL; dosage of catalyst, 0.06 g; reaction temperature, 85 °C; and reaction time, 30 min. The water-soluble chitosan with a viscosity-average molecular weight (M_v) of 4900 was obtained under the optimum degradation conditions and was characterized by FTIR, ultraviolet-visible diffuse reflection spectra (UV-vis DRS), and X-ray powder diffraction analysis.     

Interaction between tyrosineZ and substrate water in active photosystem II

In the field of photosynthetic water oxidation it has been under debate whether Tyrosine_Z (Tyr_Z) acts as a hydrogen or an electron acceptor from water. In the former concept, direct contact of Tyr_Z with substrate water has been assumed. However, there is no direct evidence for the interaction between Tyr_Z and substrate water in active Photosystem II (PSII), instead most experiments have been performed on inhibited PSII. Here, this problem is tackled in active PSII by combining low temperature EPR measurements and quantum chemistry calculations. EPR measurements observed that the maximum yield of Tyr_Z oxidation at cryogenic temperature in the S₀ and S₁ states was around neutral pH and was essentially pH-independent. The yield of Tyr_Z oxidation decreased at acidic and alkaline pH, with pKs at 4.7-4.9 and 7.7, respectively. The observed pH-dependent parts at low and high values of pH can be explained as due to sample inactivation, rather than active PSII. The reduction kinetics of Tyr_Z^· in the S₀ and S₁ states were pH independent at pH range from 4.5 to 8. Therefore, the change of the pH in bulk solution probably has no effect on the Tyr_Z oxidation and Tyr_Z^· reduction at cryogenic temperature in the S₀ and S₁ states of the active PSII. Theoretical calculations indicate that Tyr_Z becomes more difficult to oxidize when a H₂O molecule interacts directly with it. It is suggested that Tyr_Z is probably located in a hydrophobic environment with no direct interaction with the substrate H₂O in active PSII. These results provide new insights on the function and mechanism of water oxidation in PSII.     

Photosystem II heterogeneity: the acceptor side

It is well known that two photosystems, I and II, are needed to transfer electrons from H₂O to NADP⁺ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS II and PS II units, (b) RC complex containing electron acceptor Q₁ or Q₂; and (c) electron acceptor complex: Q_A (having two different redox potentials Q_L and Q_H) and Q_B (Q_B-type; Q'_B type; and non-Q_B type); additional components such as iron (Q-400), U (E_m,7=–450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.Abbreviations DCBQ 2,5 or 2,6 dichloro-p-benzoquinone - DMQ dimethylquinone - Q_A primary plastoquinone electron acceptor of photosystem II - Q_B secondary plastoquinone electron acceptor of photosystem II - IODP successive fluorescence levels during time course of chlorophyll a fluorescence: O for origin, I for inflection, D for dip or plateau, and P for peak     

Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles

Replacing the naphthalene C-unit of the anti-tuberculosis drug bedaquiline with a range of bicyclic heterocycles of widely differing lipophilicity gave analogs with a 4.5-fold range in clogP values. The biological results for these compounds indicate on average a lower clogP limit of about 5.0 in this series for retention of potent inhibitory activity (MIC₉₀s) against M.tb in culture. Some of the compounds also showed a significant reduction in inhibition of hERG channel potassium current compared with bedaquiline, but there was no common structural feature that distinguished these.     

用于超声造影的微囊藻气囊提取新方法

气囊是一种由蛋白质外壳包裹气体形成的纳米级细胞结构,常见于蓝藻和嗜盐古菌中.气囊中包含的气体在超声波作用下,能够散射声波并产生谐波信号而增强信噪比,具备成为新型超声造影剂的潜质.但目前提取气囊的方法主要是传统的高渗裂解法,该操作过程烦琐、得率低,不适用于气囊的大规模提取.针对这些技术瓶颈,文中建立了一种快速、高效的微囊...