Hormonal control of sterol biosynthesis in Phaseolus aureus

Naphthalene acetic acid increased the sterol content of mung bean hypocotyl sections mainly in the zone of elongation growth. The increased sterol synthesis can be explained by a stimulated conversion rate of cycloartenol into sterols. During the 20-hr incubation period the stigmasterol: sitosterol ratio increased considerably.     

增温、刈割对高寒草甸植被物种多样性和地下生物量的影响

我国高寒草甸表现出退化现象,严重影响着植被物种多样性和生物量生产。本研究以青藏高原高寒草甸为研究区,采用随机区组设计,设置对照、增温、刈割、增温+刈割交互作用4种试验样地,于植被生长季进行植被物种多样性(Margalef指数、Shannon指数、Simpson指数、Pielou指数)和地下生物量的调查,研究增温、刈割对高寒草甸植被物种多样性和地下生物量的影响。结果表明:(1)高寒草甸植被物种多样性在生长季中期(6、7、8月)显著高于初期(5月)和末期(9月),且显著性从2012年到2013年增强。(2)植被物种多样性对增温(3年)、刈割(2年)的响应并不敏感,在增温、刈割单独作用下略有增加,在增温+刈割交互作用下略有减小。(3)增温、刈割趋于增加地下生物量,促使地下生物量在不同土层的分配比例发生变化;随着时间延长,刈割对土壤浅层(0~30 cm土层)地下生物量的影响逐渐加强,而增温对土壤深层(30~50 cm土层)地下生物量的影响逐渐加强。     

The Ins and Outs of Ring-Cleaving Dioxygenases

ABSTRACT

Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.     

A RECOMBINANT Escherichia coli BIOSENSOR FOR DETECTING POLYCYCLIC AROMATIC HYDROCARBONS IN GAS AND AQUEOUS PHASES

Recombinant microbial biosensors are known to be simple, cheap, and very efficient monitoring tools for detecting various environmental pollutants in the field. However, although various recombinant microbial biosensors have been developed for aqueous-phase samples, very few are applicable to the gas phase. Here, we report a recombinant Escherichia coli biosensor that can be used to monitor polycyclic aromatic hydrocarbons (PAHs) in both gas and aqueous phases by color development. Among the PAHs, naphthalene and salicylate are often used as model compounds, since they are less toxic than other options and they are widely used in various applications. Here, recombinant E. coli cells carrying nahR (encoding the NahR regulatory protein for naphthalene degradation)::lac Z fusion genes were constructed and suspended (for aqueous measurements) or co-immobilized (for gaseous measurements) with chlorophenol red-ß-D-galactopyranoside (CPRG). Biosensing was then performed by ß-galactosidase, which hydrolyzed CPRG as a substrate, developing detectable red color with the naked eye. The system showed selective responses to salicylate and naphthalene. Importantly, its response to naphthalene was much more sensitive (about 10⁵-fold) in the gas phase compared to the aqueous phase. Thus, this system could potentially be used for the instrument-free, color-change-based monitoring of gaseous pollutants.     

Ascorbate-induced generation of 5-hydroxymethylcytosine is unaffected by varying levels of iron and 2-oxoglutarate

Tet (ten–eleven translocation) methylcytosine dioxygenases, which belong to the iron and 2-oxoglutarate (2OG)-dependent dioxygenase superfamily, convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. We recently reported that ascorbate (vitamin C) induces Tet-mediated generation of 5hmC. To initially delineate the role of ascorbate on 5hmC generation, we analyzed whether the effect of ascorbate is dependent upon the conditions of other components involved in the hydroxylation of 5mC catalyzed by Tet. We found that removing iron from the culture medium did not affect the induction of 5hmC by ascorbate (10 μM) in mouse embryonic fibroblasts (MEFs). The effect of ascorbate did not involve an increased expression of Tet1–3 or isocitrate dehydrogenases (IDH1–2), the enzymes responsible for producing 2OG. Interestingly, MEFs cultured with different concentrations of glucose, a major precursor of 2OG, exhibited nearly identical responses to ascorbate treatment. Further, blocking the uptake of the reduced form of vitamin C, ascorbic acid, through the sodium-dependent vitamin C transporters (SVCTs) inhibited the effect of ascorbate on 5hmC. However, inhibition of the facilitative glucose transporters (GLUTs), which mediate the incorporation of the oxidized form of vitamin C, dehydroascorbic acid (DHA), did not modify the ability of ascorbate to induce 5hmC generation. These results indicate that the effect of ascorbate on 5hmC is not dependent upon iron uptake, the expression of Tet and IDH, or the production of 2OG, suggesting that ascorbate may directly participate in the generation of 5hmC, most likely as a cofactor of Tet.     

Age‐dependent alterations of the kynurenine pathway in the YAC128 mouse model of Huntington disease

Indoleamine 2,3 dioxygenase (Ido1), the first and rate‐limiting enzyme of the kynurenine pathway (KP), is a striatally enriched gene with increased expression levels in the YAC128 mouse model of Huntington disease (HD). Our objective in this study was to delineate age‐related KP alterations in this model. Three enzymes potentially catalyze the first step of the KP; Ido1 and Indoleamine 2,3 dioxygenase‐2 were highly expressed in the striatum and Tryptophan 2,3 dioxygenase (Tdo2) in the cerebellum. During development, Ido1 mRNA expression is dynamically regulated and chronically up‐regulated in YAC128 mice. Kynurenine (Kyn) to tryptophan (Trp) ratio, a measure of activity in the first step of the KP, was elevated in YAC128 striatum, but no change in Tdo2 mRNA levels or Kyn to Trp ratio was detected in the cerebellum. Ido1 induction was coincident with Trp depletion at 3 months and Kyn accumulation at 12 months of age in striatum. Changes in downstream KP metabolites of YAC128 mice generally followed a biphasic pattern with neurotoxic metabolites reduced at 3 months and increased at 12 months of age. Striatally specific induction of Ido1 and downstream KP alterations suggest involvement in HD pathogenesis, and should be taken into account in future therapeutic developments for HD.     

Insight into the metabolism of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by biphenyl dioxygenases

In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE_LB400) and of Pandoraea pnomenusa B356 (BphAE_B356) to metabolize DDT. Data show BphAE_LB400 is unable to metabolize this substrate but BphAE_B356 metabolizes DDT to produce two stereoisomers. Structural analysis of DDT-docked BphAE_LB400 and BphAE_B356 identified residue Phe336 of BphAE_LB400 as critical to prevent productive binding of DDT to BphAE_LB400. Furthermore, the fact that residue Gly319 of BphAE_B356 is less constrained than Gly321 of BphAE_LB400 most likely contributes to the ability of BphAE_B356 to bind DDT productively. This was confirmed by examining the ability of BphAE chimeras obtained by shuffling bphA genes from strain B356 and LB400. Chimeras where residues Thr335 (which modulates the constraints on Gly321) and Phe336 (which contacts the substrate) of BphAE_LB400 were replaced by Gly and Ile respectively were able to metabolize DDT. However their stereospecificities varied depending on the presence of other segments or residues from BphAE_B356. Structural analysis suggests that either one or both of residue 267 and a segments comprised of residue 247–260 are likely involved in stereospecificity.     

Isolation and Identification of Fructo-oligosaccharides in Roots of Asparagus (Asparagus officinalis L.)

Eight fructo-oligosaccharides were isolated from purified oligosaccharide fractions of the roots of Asparagus officinalis L. (Liliaceae). By examination of constituent sugars, gas-liquid-chromatographic analysis of methyl derivatives, and investigation of partial acid hydrolyzates and products of β-fructofuranosidase action, they were confirmed to be 1^F(1-β -fructofuranosyl)_n sucrose [n = 1 (1-kestose), 2 (nystose), and 3], 6^G (1-β-fructofuranosyl)_n sucrose [n = 1 (neokestose), 2, and 3], 1^F,6^G-di-β-fructofuranosyl sucrose, and a new pentasaccharide 1^F (1-β-fructofuranosyl)₂-6^G-β-fructofuranosyl sucrose.