Improving the phenotype predictions of a yeast genome‐scale metabolic model by incorporating enzymatic constraints

Genome‐scale metabolic models (GEMs) are widely used to calculate metabolic phenotypes. They rely on defining a set of constraints, the most common of which is that the production of metabolites and/or growth are limited by the carbon source uptake rate. However, enzyme abundances and kinetics, which act as limitations on metabolic fluxes, are not taken into account. Here, we present GECKO, a method that enhances a GEM to account for enzymes as part of reactions, thereby ensuring that each metabolic flux does not exceed its maximum capacity, equal to the product of the enzyme's abundance and turnover number. We applied GECKO to a Saccharomyces cerevisiae GEM and demonstrated that the new model could correctly describe phenotypes that the previous model could not, particularly under high enzymatic pressure conditions, such as yeast growing on different carbon sources in excess, coping with stress, or overexpressing a specific pathway. GECKO also allows to directly integrate quantitative proteomics data; by doing so, we significantly reduced flux variability of the model, in over 60% of metabolic reactions. Additionally, the model gives insight into the distribution of enzyme usage between and within metabolic pathways. The developed method and model are expected to increase the use of model‐based design in metabolic engineering.     

Effect of some heavy metal ions on copper-induced metallothionein synthesis in the yeast Saccharomyces cerevisiae

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu²⁺ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd²⁺ and Mn²⁺. By adding Cd²⁺ and Mn²⁺ at 10 M concentration, the -galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO₄, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu²⁺ and 1 M-Cd²⁺. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd²⁺ environment. In contrast, the presence of Mn²⁺ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu²⁺. The recovery from growth inhibition by Mn²⁺ was due to decreased Cu²⁺ accumulation. Inhibitory concentrations of Co²⁺, Ni²⁺ and Zn²⁺ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd²⁺ and Mn²⁺. These results are discussed in relation to Cu²⁺ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.     

Fluorescent sensors based on quinoline‐containing styrylcyanine: determination of ferric ions,hydrogen peroxide,and glucose,pH‐sensitive properties and bioimaging

A novel styrylcyanine‐based fluorescent probe 1 was designed and synthesized via facile methods. Ferric ions quenched the fluorescence of probe 1, whereas the addition of ferrous ions led to only small changes in the fluorescence signal. When hydrogen peroxide was introduced into the solution containing probe 1 and Fe²⁺, Fe²⁺ was oxidized to Fe³⁺, resulting in the quenching of the fluorescence. The probe 1/Fe²⁺ solution fluorescence could also be quenched by H₂O₂ released from glucose oxidation by glucose oxidase (GOD), which means that probe 1/Fe²⁺ platform could be used to detect glucose. Probe 1 is fluorescent in basic and neutral media but almost non‐fluorescent in strong acidic environments. Such behaviour enables it to work as a fluorescent pH sensor in both the solution and solid states and as a chemosensor for detecting volatile organic compounds with high acidity and basicity. Subsequently, the fluorescence microscopic images of probe 1 in live cells and in zebrafish were achieved successfully, suggesting that the probe has good cell membrane permeability and a potential application for imaging in living cells and living organisms. Copyright © 2014 John Wiley & Sons, Ltd.     

Strong dominance of functional alleles over gene deletions in both intensely growing and deeply starved yeast cells

Previous studies with diploid yeast have shown that the deletion of one allele at a single locus typically has little impact on fitness under conditions promoting fast growth. Here, we confirm and quantify this finding. The strong dominance of functional over nonfunctional alleles is predicted by the metabolic control theory which assumes that the cell is a system of metabolic fluxes and that the total metabolic rate is equivalent to fitness. To test whether these requirements are critical, we tested dominance under conditions of long‐term starvation when metabolism is low and thus the metabolic activities of proteins are likely inadequate or imbalanced. More fundamentally, the central assumption of the model, that high metabolic rate translates into high fitness, appears implausible. Contrary to these conjectures, we found that the mean rate of survival of starving heterozygotes was affected only slightly more than was the mean rate of growth under good conditions. Under none of the two treatments the central prediction of the model, that fitness of heterozygous strains is higher for the enzymatic proteins than for nonenzymatic ones, was confirmed. Our data add to growing uncertainty whether the metabolic control theory is sufficient to explain the remarkable ubiquity of strong genetic dominance.     

The stimulatory effect of negative air ions and hydrogen peroxide on the activity of superoxide dismutase

The activity of erythrocyte cytosolic superoxide dismutase from rat, bovine, man and duck was considerably increased when measured after preparation or incubation in media pretreated with negative air ions (mostly superoxide) from electroeffluvial ion generator. 0.5–1.0 μM H₂O₂ was found in incubation medium after treatment with air ions. The stimulatory effect of air ions on superoxide dismutase activity was mimicked by addition of 0.5–6 μM H₂O₂. The primary physicochemical mechanism of beneficial biological action of negative air ions is suggested to be related to the stimulation of superoxide dismutase activity by micromolar concentrations of H₂O₂.     

Effects of Gas6 and hydrogen peroxide in Axl ubiquitination and downregulation

The receptor tyrosine kinase Axl has been shown to be activated by its ligand Gas6 and by oxidative stress in the form of hydrogen peroxide. However, the regulatory mechanisms controlling the levels of Axl upon Gas6 binding or oxidative stress have not been elucidated. This report demonstrates that Gas6-induced downregulation of Axl is blocked by inhibitors of endocytosis and lysosomal degradation, but not by inhibitors of proteosomal activity. Furthermore, it is shown that binding of Axl to Gas6 induces the phosphorylation and ubiquitination of Axl and the interaction of Axl with the ubiquitin ligase c-Cbl. Importantly, hydrogen peroxide induces Axl tyrosine phosphorylation but not its ubiquitination, determining the inhibition of Axl downregulation. These results suggest that as shown for other receptor tyrosine kinases, ubiquitination of Axl is needed to ensure its proper degradation in the lysosome, and that oxidative stress may inhibit Axl ubiquitination and downregulation.     

Effects of chloroperoxidase and hydrogen peroxide on the viabilities of Aspergillus flavus conidiospores

The effects of chloroperoxidase [EC 1.1.1.10] and hydrogen peroxide on the viabilities of quiescent and germinating conidiospores of an aflatoxigenic fungus, Aspergillus flavus, were determined. Hydrogen peroxide was found moderately lethal and chloroperoxidase produced a 30-fold increase in the lethality of hydrogen peroxide to germinating conidia, which were 75-fold more susceptible to chloroperoxidase than were quiescent conidia. According to infrared examinations of fungal corpses, mortality occurred by oxidation rather than peroxidative chlorination.     

Influence of gene position on the expression divergence of oxidative response genes in intraspecific yeast

Phenotypic variation can arise from differences in the protein coding sequence and in the regulatory elements. However, little is known about the contribution of regulatory difference to the expression divergence, especially the cis and trans regulatory variation to the expression divergence in intraspecific populations. In this study, we used two different yeast strains, BY4743 and RM11‐1a/α, to study the regulatory variation to the expression divergence between BY and RM under oxidative stress condition. Our results indicated that the expression divergence of BY and RM is mainly due to trans regulatory variations under both normal and oxidative stress conditions. However, cis regulatory variation seems to play a very important role in oxidative stress response in yeast because 36% of genes showed an increase in cis regulatory variation effect compared with 13% of genes that showed an increase in trans regulatory variation effect after oxidative stress. Our data also indicated that genes located on the longer arm of the chromosomes are more susceptible to cis variation effect under oxidative stress than genes on the shorter arm of the chromosomes.     

Effects of destruxins on free calcium and hydrogen ions in insect hemocytes

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the ento mopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects ofdestruxins on changes in free calcium and hydrogen ions in the hemocytes ofExolontha serrulata, Bombyx mori and the Spodoptera litura SL1 cell line were detected using laser scanning confocal mi croscopy （LSCM）. An instant Ca2＋ influx of hemocytes induced by destruxins A and B （DA and DB） was recorded. The DA/DBdependent Ca2＋ influx was not influenced by the Ca2＋ channel inhibitors 2aminoethoxydiphenyl borane （2APB） and U73122. It also had an apparently different LSCM profile from that of the ionomycindependent Ca2＋ influx. However, the instant Ca2＋ influx was not seen in the SL1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2＋ was observed. Meanwhile, an instant intracellular free H＋ decrease aroused by DA and DB was found. DB at 20/zmol/L and DA at 690/zmol/L significantly reduced intracellular free H＋ levels. Furthermore, the vacuolar H＋ATPase （VATPase） inhibitor bafilomycin A1 had obvious effects on the decreases ofintracellular free H＋ in hemocytes. These results suggest that the mechanism of DA/DBdependent Ca2＋ influx is perhaps not related to Ca2＋ channels and ionophores; rather, the intracellular free H＋ decrease might be due to VATPase inhibition.     

过氧化氢预处理对褐煤物化性质及生物产气的影响

【目的】研究过氧化氢预处理对褐煤物化性质及生物产气的影响。【方法】以胜利5号褐煤为研究对象,利用正交试验对过氧化氢预处理褐煤条件进行优化,在最优条件下处理褐煤得到处理后的残煤和处理液,通过X射线衍射分析(X-ray diffraction, XRD)、扫描电镜分析(scanning electron microscopy, SEM)、比表面积分析及孔隙分析(brunauer-emmett-teller, BET)、气相色谱-质谱分析(GC-MS)、高效液相色谱分析(HPLC)等方法对原煤、残煤和处理液的物化性质进行比较分析。【结果】经过氧化氢预处理,褐煤的最优条件为过氧化氢浓度5.0%、预处理时间20 d、液固比30:1,处理液中总有机碳含量为105 mg/L。在最优条件下,过氧化氢处理后残煤表面裂痕、凹陷增多,表面结构变得松散;煤的芳香面网间距增加,芳环结构更加疏松,晶核结构变小;孔隙度和比表面积均增大。处理后残煤中的固定碳、C元素和镜质组的相对含量降低,而灰分、挥发分、O和H元素及惰质组含量增加,残煤中O=C-O、C=C、C=O官能团含量增加,而N-H、C-H官能团含量则减少。生物产气结果表明反应液和残煤产气量均低于原煤,分别减少了39.13%和94.46%。过氧化氢预处理主要作用于煤中镜质组,使其有机碳溶解,煤中大分子结构的官能团发生变化,改变煤的芳环结构,在氧化作用下煤结构中的小分子溶解进入处理液。处理液中有机物以短链脂肪酸为主。经生物产气后,反应液中小分子酸以及有机物种类减少,被微生物利用产气。而各产气试验组中优势菌门及优势菌属的菌群丰度呈现出显著差异,古菌中原煤产气组盐杆菌门(Halobacteriota)为优势菌门,甲烷八叠球菌属(Methanosarcina)为优势菌属;反应液产气组热变形菌(Thermoprotei)为优势菌门,深古菌属(Bathyarchaeia)为优势菌属;细菌中原煤产气组放线菌门(Actinomycetota)为优势菌门,Gaiellales为优势菌属;反应液产气试验组假单胞菌门(Pseudomonadota)为优势菌门,代尔夫特菌属(Delftia)为优势菌属。【结论】煤溶解有机碳可以被微生物利用产气,但是煤中有机组分的过氧化脱除导致生物产气量减少。     

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid‐mediated systemic virus resistance in Nicotiana benthamiana

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H₂O₂ and NO. Scavenging of H₂O₂ or NO in upper leaves blocked BR‐induced systemic virus resistance. BR‐induced systemic H₂O₂ accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite‐dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR‐triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR‐activated H₂O₂ is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H₂O₂ generation blocked BR‐induced systemic NO production, but BR‐induced H₂O₂ production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR‐induced systemic virus defense in NbRBOHB‐silenced plants, but H₂O₂ did not reverse the effect of NbNR silencing on BR‐induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR‐mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR‐induced H₂O₂ and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR‐mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)‐dependent H₂O₂ production and subsequent systemic NR‐dependent NO generation.     

Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.     

Effects of methylglyoxal on platelet hydrogen peroxide accumulation,aggregation and release reaction

Methylglyoxal generates a slight increase in the basal level of hydrogen peroxide in platelets. The oxidation effect of methylglyoxal significantly potentiated by thrombin, depends on both the ketoaldehyde and the agonist concentrations. A further significant increase in hydrogen peroxide accumulation was obtained in platelets pretreated with the alkylating agent N-ethylmaleimide which depletes GSH and blocks glutathione peroxidase. Resting platelets completely transform the ketoaldehyde into D (?)lactate, whereas stimulated platelets transform about 10–15 per cent of the metabolized methylglyoxal into D (?)lactate. The metabolic modifications generated by methylglyoxal such as the GSH depletion and hydrogen peroxide accumulation induce modifications in platelet function. Methylglyoxal inhibits platelet aggregation induced by several agonists and ATP release induced by thrombin.     

Effect of hydrogen peroxide on the activity and structure of Escherichia coli chaperone GroEL

Chaperone GroEL was treated with different concentrations of hydrogen peroxide. The conformational states of GroEL were monitored by protein intrinsic fluorescence, 8-anilino-1-naphthalene sulfonate fluorescence, and far-UV CD measurements. The results show that GroEL has unusual ability to resist oxidative stress. GroEL kept its quaternary structure and activity even when treated with 10 mM hydrogen peroxide. Two fragments were formed when GroEL was treated with high concentrations of hydrogen peroxide (more than 20 mM). It is suggested that GroEL, as a molecular chaperone, is related to oxidative process in vivo.