H2S and its role in redox signaling

Hydrogen sulfide (H₂S) has emerged as an important gaseous signaling molecule that is produced endogenously by enzymes in the sulfur metabolic network. H₂S exerts its effects on multiple physiological processes important under both normal and pathological conditions. These functions include neuromodulation, regulation of blood pressure and cardiac function, inflammation, cellular energetics and apoptosis. Despite the recognition of its biological importance and its beneficial effects, the mechanism of H₂S action and the regulation of its tissue levels remain unclear in part owing to its chemical and physical properties that render handling and analysis challenging. Furthermore, the multitude of potential H₂S effects has made it difficult to dissect its signaling mechanism and to identify specific targets. In this review, we focus on H₂S metabolism and provide an overview of the recent literature that sheds some light on its mechanism of action in cellular redox signaling in health and disease. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Folding of AcrB Subunit Precedes Trimerization

AcrB and its homologues are major players in the efflux of anti-microbials out of Gram-negative bacteria. The structural and functional unit of AcrB is a homo-trimer. The assembly process of obligate membrane protein oligomers, including AcrB, remains elusive. It is not clear if an individual subunit folds into a monomeric form first followed by association (three-stage pathway) or if association occurs simultaneously with subunit folding (two-stage pathway). To answer this question, we investigated the feasibility of creating a folded monomeric AcrB mutant. The existence of well-folded monomers in the cell membrane would be an evidence of a three-stage pathway. A monomeric AcrB mutant, AcrB_Δloop, was created through the truncation of a protruding loop that appeared to contribute to the stability of an AcrB trimer. AcrB_Δloop expressed at a level similar to that of wild-type AcrB. The secondary structure content and tertiary conformation of AcrB_Δloop were very similar to those of wild-type AcrB. However, when expressed in an acrB-deficient strain, AcrB_Δloop failed to complement its defect in drug efflux. Results from blue native polyacrylamide gel electrophoresis and chemical cross-linking experiments suggested that AcrB_Δloop existed as a monomer. The expression of this monomeric mutant in a wild-type Escherichia coli strain did not have a significant dominant-negative effect, suggesting that the mutant could not effectively co-assemble with genomic AcrB. AcrB_Δloop is the first monomeric mutant reported for the intrinsically trimeric AcrB. The structural characterization results of this mutant suggest that the oligomerization of AcrB occurs through a three-stage pathway involving folded monomers.     

Advances in thermochemical conversion of woody biomass to energy,fuels and chemicals

Biomass has been recognised as a promising resource for future energy and fuels. The biomass, originated from plants, is renewable and application of its derived energy and fuels is close to carbon-neutral by considering that the growing plants absorb CO₂ for photosynthesis. However, the complex physical structure and chemical composition of the biomass significantly hinder its conversion to gaseous and liquid fuels.This paper reviews recent advances in biomass thermochemical conversion technologies for energy, liquid fuels and chemicals. Combustion process produces heat or heat and power from the biomass through oxidation reactions; however, this is a mature technology and has been successfully applied in industry. Therefore, this review will focus on the remaining three thermochemical processes, namely biomass pyrolysis, biomass thermal liquefaction and biomass gasification. For biomass pyrolysis, biomass pretreatment and application of catalysts can simplify the bio-oil composition and retain high yield. In biomass liquefaction, application of appropriate solvents and catalysts improves the liquid product quality and yield. Gaseous product from biomass gasification is relatively simple and can be further processed for useful products. Dual fluidised bed (DFB) gasification technology using steam as gasification agent provides an opportunity for achieving high hydrogen content and CO₂ capture with application of appropriate catalytic bed materials. In addition, multi-staged gasification technology, and integrated biomass pyrolysis and gasification as well as gasification for poly-generation have attracted increasing attention.     

Order-disorder-order transitions mediate the activation of cholera toxin

Cholera toxin (CT) holotoxin must be activated to intoxicate host cells. This process requires the intracellular dissociation of the enzymatic CTA1 domain from the holotoxin components CTA2 and B5, followed by subsequent interaction with the host factor ADP ribosylation factor 6 (ARF6)-GTP. We report the first NMR-based solution structural data for the CT enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus and binds its protein partners at both the beginning and the end of this activation process. Deviations from random coil chemical shifts (Δδ_coil) indicate helix formation in the activation loop, which is essential to open the toxin's active site and occurs prior to its association with human protein ARF6. We performed NMR titrations of both free CTA1 and an active CTA1:ARF6-GTP complex with NAD⁺, which revealed that the formation of the complex does not significantly enhance NAD⁺ binding. Partial unfolding of CTA1 is further illustrated by using 4,4′-bis(1-anilinonaphthalene 8-sulfonate) fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP. We propose that the primary role of ARF6's allostery is to induce refolding of the C-terminus of CTA1. Thus, as a folded globular toxin complex, CTA1 escapes the chaperone and proteasomal components of the endoplasmic reticulum associated degradation pathway in the cytosol and then proceeds to ADP ribosylate its target G_sα, triggering the downstream events associated with the pathophysiology of cholera.     

Chemical fingerprint,acute oral toxicity and anti-inflammatory activity of the hydroalcoholic extract of leaves from Tocoyena formosa (Cham. & Schlecht.) K. Schum

Inflammation is a protective response of the organism against damaging agents, this process is considered beneficial, however in some situations, this response can be damage when exacerbated effect are present. This claim objective to evaluate the qualitative and quantitative chemical profile, acute toxic and anti-inflammatory effects of the hydroalcoholic extract of leaves from Tocoyena formosa (Cham. & Schlecht.) K. Schum. (HELTF). Quantitative and qualitative phytochemical analysis was performed by HPLC-DAD and colorimetric assay. The topical anti-inflammatory activity was determined in Croton oil-induced ear edema assay and systemic activity was performed in vascular permeability, paw edema induced by carrageenan and dextran. Phytochemical analysis of leaves from HELTF showed presence of tannin, flavonoid, saponins an other that confirmed by HPLC analysis. The extract did not cause significant with LD₅₀ greater than 5000 mg/kg and did not promote significate reduction in topical inflammatory process. However, HELTF demonstrate significant reduction of paw edema induced by carrageenan and dextran. The HELTF (200 mg/kg) reduced the protein/cell migration in the intradermal carrageenan-induced inflammation. Our results demonstrated that the first time the chemical profile and describe the effective action in systemic anti-inflammatory, antiedematogenic activity and low acute toxicity. This activity presents, supporting its traditional use. However, new studies are necessary for the detection and clarification of the possible mechanism of action.     

The Role of the Electrode Surface in Na–Air Batteries: Insights in Electrochemical Product Formation and Chemical Growth of NaO2

The Na–air battery, because of its high energy density and low charging overpotential, is a promising candidate for low‐cost energy storage, hence leading to intensive research. However, to achieve such a battery, the role of the positive electrode material in the discharge process must be understood. This issue is herein addressed by exploring the electrochemical reduction of oxygen, as well as the chemical formation and precipitation of NaO₂ using different electrodes. Whereas a minor influence of the electrode surface is demonstrated on the electrochemical formation of NaO₂, a strong dependence of the subsequent chemical precipitation of NaO₂ is identified. In the origin, this effect stems from the surface energy and O₂/O₂^? affinity of the electrode. The strong interaction of Au with O₂/O₂^? increases the nucleation rate and leads to an altered growth process when compared to C surfaces. Consequently, thin (3 µm) flakes of NaO₂ are found on Au, whereas on C large cubes (10 µm) of NaO₂ are formed. This has significant impact on the cell performance and leads to four times higher capacity when C electrodes with low surface energy and O₂/O₂^? affinity are used. It is hoped that these findings will enable the design of new positive electrode materials with optimized surfaces.     

Structural Insights into Regioselectivity in the Enzymatic Chlorination of Tryptophan

The regioselectively controlled introduction of chlorine into organic molecules is an important biological and chemical process. This importance derives from the observation that many pharmaceutically active natural products contain a chlorine atom. Flavin-dependent halogenases are one of the principal enzyme families responsible for regioselective halogenation of natural products. Structural studies of two flavin-dependent tryptophan 7-halogenases (PrnA and RebH) have generated important insights into the chemical mechanism of halogenation by this enzyme family. These proteins comprise two modules: a flavin adenine dinucleotide (FAD)-binding module and a tryptophan-binding module. Although the 7-halogenase studies advance a hypothesis for regioselectivity, this has never been experimentally demonstrated. PyrH is a tryptophan 5-halogenase that catalyzes halogenation on tryptophan C5 position. We report the crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD. The FAD-binding module is essentially unchanged relative to PrnA (and RebH), and PyrH would appear to generate the same reactive species from Cl⁻, O₂, and 1,5-dihydroflavin adenine dinucleotide. We report additional mutagenesis data that extend our mechanistic understanding of this process, in particular highlighting a strap region that regulates FAD binding, and may allow communication between the two modules. PyrH has a significantly different tryptophan-binding module. The data show that PyrH binds tryptophan and presents the C5 atom to the reactive chlorinating species, shielding other potential reactive sites. We have mutated residues identified by structural analysis as recognizing the tryptophan in order to confirm their role. This work establishes the method by which flavin-dependent tryptophan halogenases regioselectively control chlorine addition to tryptophan. This method would seem to be general across the superfamily.     

综述与专论: 贵金属和碳纳米酶分子机制的理论研究

2007年四氧化三铁类过氧化物酶活性的发现催生了纳米酶这一新兴多学科交叉研究方向,多种基于金属、金属氧化物和碳纳米材料的纳米酶被发现,并在环境,食品安全,化工,生物医学等领域获得应用。相应的,纳米酶催化分子机制的理论研究也取得了进展。本文将回顾化学催化的基本原理,重点总结贵金属和碳纳米酶分子机制的理论研究进展。     

Phenotypic Plasticity Contributes to Maize Adaptation and Heterosis

Plant phenotypic plasticity describes altered phenotypic performance of an individual when grown in different environments. Exploring genetic architecture underlying plant plasticity variation may help mitigate the detrimental effects of a rapidly changing climate on agriculture, but little research has been done in this area to date. In the present study, we established a population of 976 maize F₁ hybrids by crossing 488 diverse inbred lines with two elite testers. Genome-wide association study identified hundreds of quantitative trait loci associated with phenotypic plasticity variation across diverse F₁ hybrids, the majority of which contributed very little variance, in accordance with the polygenic nature of these traits. We identified several quantitative trait locus regions that may have been selected during the tropical-temperate adaptation process. We also observed heterosis in terms of phenotypic plasticity, in addition to the traditional genetic value differences measured between hybrid and inbred lines, and the pattern of which was affected by genetic background. Our results demonstrate a landscape of phenotypic plasticity in maize, which will aid in the understanding of its genetic architecture, its contribution to adaptation and heterosis, and how it may be exploited for future maize breeding in a rapidly changing environment.     

The distribution of residues in a polypeptide sequence is a determinant of aggregation optimized by evolution

It has been shown that the propensity of a protein to form amyloid-like fibrils can be predicted with high accuracy from the knowledge of its amino acid sequence. It has also been suggested, however, that some regions of the sequences are more important than others in determining the aggregation process. Here, we have addressed this issue by constructing a set of “sequence scrambled” variants of the first 29 residues of horse heart apomyoglobin (apoMb_1-29), in which the sequence was modified while maintaining the same amino acid composition. The clustering of the most amyloidogenic residues in one region of the sequence was found to cause a marked increase of the elongation rate (k_agg) and a remarkable shortening of the lag phase (t_lag) of the fibril growth, as determined by far-UV circular dichroism and thioflavin T fluorescence. We also show that taking explicitly into consideration the presence of aggregation-promoting regions in the predictive methods results in a quantitative agreement between the theoretical and observed k_agg and t_lag values of the apoMb_1-29 variants. These results, together with a comparison between homologous segments from the family of globins, indicate the existence of a negative selection against the clustering of highly amyloidogenic residues in one or few regions of polypeptide sequences.     

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants

Iron (Fe) is essential for virtually all living organisms. The identification of the chemical forms of iron (the speciation) circulating in and between cells is crucial to further understand the mechanisms of iron delivery to its final targets. Here we analyzed how iron is transported to the seeds by the chemical identification of iron complexes that are delivered to embryos, followed by the biochemical characterization of the transport of these complexes by the embryo, using the pea (Pisum sativum) as a model species. We have found that iron circulates as ferric complexes with citrate and malate (Fe(III)₃Cit₂Mal₂, Fe(III)₃Cit₃Mal₁, Fe(III)Cit₂). Because dicotyledonous plants only transport ferrous iron, we checked whether embryos were capable of reducing iron of these complexes. Indeed, embryos did express a constitutively high ferric reduction activity. Surprisingly, iron(III) reduction is not catalyzed by the expected membrane-bound ferric reductase. Instead, embryos efflux high amounts of ascorbate that chemically reduce iron(III) from citrate-malate complexes. In vitro transport experiments on isolated embryos using radiolabeled ⁵⁵Fe demonstrated that this ascorbate-mediated reduction is an obligatory step for the uptake of iron(II). Moreover, the ascorbate efflux activity was also measured in Arabidopsis embryos, suggesting that this new iron transport system may be generic to dicotyledonous plants. Finally, in embryos of the ascorbate-deficient mutants vtc2-4, vtc5-1, and vtc5-2, the reducing activity and the iron concentration were reduced significantly. Taken together, our results identified a new iron transport mechanism in plants that could play a major role to control iron loading in seeds.     

Skp1-Cul1-F-box Ubiquitin Ligase (SCFβTrCP)-mediated Destruction of the Ubiquitin-specific Protease USP37 during G2-phase Promotes Mitotic Entry

Ubiquitin-mediated proteolysis is a key regulatory process in cell cycle progression. The Skp1-Cul1-F-box (SCF) and anaphase-promoting complex (APC) ubiquitin ligases target numerous components of the cell cycle machinery for destruction. Throughout the cell cycle, these ligases cooperate to maintain precise levels of key regulatory proteins, and indirectly, each other. Recently, we have identified the deubiquitinase USP37 as a regulator of the cell cycle. USP37 expression is cell cycle-regulated, being expressed in late G₁ and ubiquitinated by APC^Cdh1 in early G₁. Here we report that in addition to destruction at G₁, a major fraction of USP37 is degraded at the G₂/M transition, prior to APC substrates and similar to SCF^βTrCP substrates. Consistent with this hypothesis, USP37 interacts with components of the SCF in a βTrCP-dependent manner. Interaction with βTrCP and subsequent degradation is phosphorylation-dependent and is mediated by the Polo-like kinase (Plk1). USP37 is stabilized in G₂ by depletion of βTrCP as well as chemical or genetic manipulation of Plk1. Similarly, mutation of the phospho-sites abolishes βTrCP binding and renders USP37 resistant to Plk1 activity. Expression of this mutant hinders the G₂/M transition. Our data demonstrate that tight regulation of USP37 levels is required for proper cell cycle progression.     

Evidence Against the “Y–T Coupling” Mechanism of Activation in the Response Regulator NtrC

The dominant theory on the mechanism of response regulators activation in two-component bacterial signaling systems is the “Y–T coupling” mechanism, wherein the χ₁ rotameric state of a highly conserved aromatic residue correlates with the activation of the protein via structural rearrangements coupled to a conserved tyrosine. In this paper, we present evidence that, in the receiver domain of the response regulator nitrogen regulatory protein C (NtrC^R), the interconversion of this tyrosine (Y101) between its rotameric states is actually faster than the rate of inactive/active conversion and is not correlated to the activation process. Data gathered from NMR relaxation dispersion experiments show that a subset of residues surrounding the conserved tyrosine sense a process that is occurring at a faster rate than the inactive/active conformational transition. We show that this process is related to χ₁ rotamer exchange of Y101 and that mutation of this aromatic residue to a leucine eliminated this second faster process without affecting activation. Computational simulations of NtrC^R in its active conformation further demonstrate that the rotameric state of Y101 is uncorrelated with the global conformational transition during activation. Moreover, the tyrosine does not appear to be involved in the stabilization of the active form upon phosphorylation and is not essential in propagating the signal downstream for ATPase activity of the central domain. Our data provide experimental evidence against the generally accepted “Y–T coupling” mechanism of activation in NtrC^R.     

THE ACTION OF INHIBITORY NERVES ON CARBON DIOXIDE PRODUCTION IN THE HEART GANGLION OF LIMULUS

It has been shown in this paper that stimulation of the inhibitory nerves of the neurogenic heart of Limulus, which correspond to the vagus nerves of the vertebrate heart, results in a marked diminution of CO₂ production in the heart ganglion, while stimulation of the ganglion, leading to increased activity of the heart, leads also to increased CO₂ production by the ganglion. This shows that inhibition of the automaticity of this ganglion by the action of its inhibitory nerves consists, not in a process of blocking, but in a diminution of those chemical reactions in the ganglion cells which give rise to the production of CO₂.     

Solution Conformation and Dynamics of the HIV-1 Integrase Core Domain

The human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a critical enzyme involved in infection. It catalyzes two reactions to integrate the viral cDNA into the host genome, 3′ processing and strand transfer, but the dynamic behavior of the active site during catalysis of these two processes remains poorly characterized. NMR spectroscopy can reveal important structural details about enzyme mechanisms, but to date the IN catalytic core domain has proven resistant to such an analysis. Here, we present the first NMR studies of a soluble variant of the catalytic core domain. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the α4 helix extends toward the active site. We also observe a dramatic improvement in NMR spectra with increasing MgCl₂ concentration. This improvement suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg²⁺. In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full-length enzyme. ¹⁵N relaxation experiments further show that, although conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters that show striking similarity to a recent crystal structure of prototype foamy virus IN bound to DNA.     

Chemical intermediates in dopamine oxidation by tyrosinase,and kinetic studies of the process

A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H⁺ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine → o-dopaminequinone-H⁺ → o-dopaminequinone → leuko-dopaminochrome → dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H⁺ was transformed into aminochrome in a constant ratio. The stoichiometry equation for this conversion is 2 o-dopaminequinone-H⁺ → dopamine + dopaminochrome. The pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named “Enzymatic-chemical-chemical” (E_ZCC). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H⁺ to dopaminequinone, and the internal cyclization of dopaminequinone to leukodopaminochrome have been calculated.     

Fenugreekine,a new steroidal sapogenin-peptide ester of Trigonella foenum-graecum

A new C₂₇-steroidal sapogenin-peptide ester, fenugreekine, has been isolated from seeds of Trigonella foenum-graecum. On acid hydrolysis, it afforded diosgenin, yamogenin, (25R)-spirosta-3,5-diene, a mixture of three isomeric (2S,3R,4R-, 2S,3R,4S-, 2S,3S,4R-)-4-hydroxyisoleucine lactones, 4′-hydroxyisoleucyl-4-hydroxyisoleucine lactone, and a C₁₄-dipeptide which was partially characterized. On the basis of this chemical transformation and spectral (UV, IR, PMR, MS) evidence of fenugreekine and its transformation products, the steroidal sapogenin-peptide ester is assigned structure (1). The two dipeptides also have not been encountered before in nature or prepared synthetically. The compound shows a number of interesting pharmacological and virological activities.     

Methane emission by plant communities in an alpine meadow on the Qinghai-Tibetan Plateau: a new experimental study of alpine meadows and oat pasture

Recently, plant-derived methane (CH₄) emission has been questioned because limited evidence of the chemical mechanism has been identified to account for the process. We conducted an experiment with four treatments (i.e. winter-grazed, natural alpine meadow; naturally restored alpine meadow eight years after cultivation; oat pasture and bare soil without roots) during the growing seasons of 2007 and 2008 to examine the question of CH₄ emission by plant communities in the alpine meadow. Each treatment consumed CH₄ in closed, opaque chambers in the field, but two types of alpine meadow vegetation reduced CH₄ consumption compared with bare soil, whereas oat pasture increased consumption. This result could imply that meadow vegetation produces CH₄. However, measurements of soil temperature and water content showed significant differences between vegetated and bare soil and appeared to explain differences in CH₄ production between treatments. Our study strongly suggests that the apparent CH₄ production by vegetation, when compared with bare soil in some previous studies, might represent differences in soil temperature and water-filled pore space and not the true vegetation sources of CH₄.