Pre-steady-state kinetic characterization of thiolate anion formation in human leukotriene C₄ synthase

Human leukotriene C? synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C? (LTC?). This molecule, together with its metabolites LTD? and LTE?, induces inflammatory responses, particularly in asthma, and thus, the enzyme is an attractive drug target. During the catalytic cycle, glutathione (GSH) is activated by hLTC4S that forms a nucleophilic thiolate anion that will attack LTA?, presumably according to an S(N)2 reaction to form LTC?. We observed that GSH thiolate anion formation is rapid and occurs at all three monomers of the homotrimer and is concomitant with stoichiometric release of protons to the medium. The pK(a) (5.9) for enzyme-bound GSH thiol and the rate of thiolate formation were determined (k(obs) = 200 s?1). Taking advantage of a strong competitive inhibitor, glutathionesulfonic acid, shown here by crystallography to bind in the same location as GSH, we determined the overall dissociation constant (K(d((GS) = 14.3 μM). The release of the thiolate was assessed using a GSH release experiment (1.3 s?1). Taken together, these data establish that thiolate anion formation in hLTC4S is not the rate-limiting step for the overall reaction of LTC? production (k(cat) = 26 s?1), and compared to the related microsomal glutathione transferase 1, which displays very slow GSH thiolate anion formation and one-third of the sites reactivity, hLTC4S has evolved a different catalytic mechanism.     

Response of Net Ecosystem Productivity of Three Boreal Forest Stands to Drought

In 2000–03, continuous eddy covariance measurements of carbon dioxide (CO₂) flux were made above mature boreal aspen, black spruce, and jack pine forests in Saskatchewan, Canada, prior to and during a 3-year drought. During the 1st drought year, ecosystem respiration (R) was reduced at the aspen site due to the drying of surface soil layers. Gross ecosystem photosynthesis (GEP) increased as a result of a warm spring and a slow decrease of deep soil moisture. These conditions resulted in the highest annual net ecosystem productivity (NEP) in the 9 years of flux measurements at this site. During 2002 and 2003, a reduction of 6% and 34% in NEP, respectively, compared to 2000 was observed as the result of reductions in both R and GEP, indicating a conservative response to the drought. Although the drought affected most of western Canada, there was considerable spatial variability in summer rainfall over the 100-km extent of the study area; summer rainfalls in 2001 and 2002 at the two conifer sites minimized the impact of the drought. In 2003, however, precipitation was similarly low at all three sites. Due to low topographic position and consequent poor drainage at the black spruce site and the coarse soil with low water-holding capacity at the jack pine site almost no reduction in R, GEP, and NEP was observed at these two sites. This study shows that the impact of drought on carbon sequestration by boreal forest ecosystems strongly depends on rainfall distribution, soil characteristics, topography, and the presence of vegetation that is well adapted to these conditions. The online version of the original article can be found under doi:     

Detection of viral sequence fragments of HIV-1 subfamilies yet unknown

Background  

Methods of determining whether or not any particular HIV-1 sequence stems - completely or in part - from some unknown HIV-1 subtype are important for the design of vaccines and molecular detection systems, as well as for epidemiological monitoring. Nevertheless, a single algorithm only, the Branching Index (BI), has been developed for this task so far. Moving along the genome of a query sequence in a sliding window, the BI computes a ratio quantifying how closely the query sequence clusters with a subtype clade. In its current version, however, the BI does not provide predicted boundaries of unknown fragments.     

Evidence for the presence of three distinct binding sites for the thioflavin T class of Alzheimer's disease PET imaging agents on beta-amyloid peptide fibrils

Imaging the progression of Alzheimer's disease would greatly facilitate the discovery of therapeutics, and a wide range of ligands are currently under development for the detection of beta-amyloid peptide (Abeta)-containing plaques by using positron emission tomography. Here we report an in-depth characterization of the binding of seven previously described ligands to in vitro generated Abeta-(1-40) polymers. All of the compounds were derived from the benzothiazole compound thioflavin T and include 2-[4'-(methylamino)phenyl]benzothiazole and 2-(4'-dimethylamino-)phenyl-imidazo[1,2-a]-pyridine derivatives, 2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole and 2-[4'-(4'-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and a benzofuran compound (5-bromo-2-(4-dimethylaminophenyl)benzofuran). By using a range of fluorescent and radioligand binding assays, we find that these compounds display a more complex binding pattern than described previously and are consistent with three classes of binding sites on the Abeta fibrils. All of the compounds bound with very high affinity (low nm K(d)) to a low capacity site (BS3) (1 ligand-binding site per approximately 300 Abeta-(1-40) monomers) consistent with the previously recognized binding site for these compounds on the fibrils. However, the compounds also bound with high affinity (K(d) approximately 100 nm) to either one of two additional binding sites on the Abeta-(1-40) polymer. The properties of these sites, BS1 and BS2, suggest they are adjacent or partially overlapping and have a higher capacity than BS3, occurring every approximately 35 or every approximately 4 monomers of Abeta-(1-40)-peptide, respectively. Compounds appear to display selectivity for BS2 based on the presence of a halogen substitution (2-[4'-(dimethylamino)phenyl]-6-iodobenzothiazole, 2-[4'-(4'-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, and 5-bromo-2-(4-dimethylaminophenyl)benzofuran) on their aromatic ring system. The presence of additional ligand-binding sites presents potential new targets for ligand development and may allow a more complete modeling of the current positron emission tomography data.     

Observation of two modes of inhibition of human microsomal prostaglandin E synthase 1 by the cyclopentenone 15-deoxy-Δ(12,14)-prostaglandin J(2)

Microsomal prostaglandin E synthase 1 (MPGES1) is an enzyme that produces the pro-inflammatory molecule prostaglandin E(2) (PGE(2)). Effective inhibitors of MPGES1 are of considerable pharmacological interest for the selective control of pain, fever, and inflammation. The isoprostane, 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a naturally occurring degradation product of prostaglandin D(2), is known to have anti-inflammatory properties. In this paper, we demonstrate that 15d-PGJ(2) can inhibit MPGES1 by covalent modification of residue C59 and by noncovalent inhibition through binding at the substrate (PGH(2)) binding site. The mechanism of inhibition is dissected by analysis of the native enzyme and the MPGES1 C59A mutant in the presence of glutathione (GSH) and glutathione sulfonate. The location of inhibitor adduction and noncovalent binding was determined by triple mass spectrometry sequencing and with backbone amide H/D exchange mass spectrometry. The kinetics, regiochemistry, and stereochemistry of the spontaneous reaction of GSH with 15d-PGJ(2) were determined. The question of whether the anti-inflammatory properties of 15d-PGJ(2) are due to inhibition of MPGES1 is discussed.     

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.     

水淹频率变化对鄱阳湖增强型植被指数的影响

水淹状况是湿地植被动态的重要影响因素。该研究基于谷歌地球引擎(GEE)平台, 利用2000-03-01至2020-02-29所有覆盖研究区域的MODIS遥感影像数据, 分析20年间水淹频率(IF)、增强型植被指数(EVI)的时空变化以及湿地植被对IF变化的响应, 得出以下结论: (1) 20年来鄱阳湖水文节律发生了明显改变, 高IF (IF > 75%)水域面积呈现下降趋势, 从2000年1 435.3 km²下降至2019年的510.25 km², 降幅为64.45%; (2)区域平均EVI呈显著上升趋势, 植被扩张主要集中在中部IF下降区域; (3)分析不同总水淹频率区域中平均EVI年际变化, 发现EVI与水淹状况的变化趋势相似, 2009年之后鄱阳湖水域面积萎缩趋势缓解, EVI增长速度出现下降; (4)鄱阳湖湿地植被主要沿水域面积萎缩方向扩张, 基于像元统计20年间IF与EVI的变化趋势, 发现它们在空间分布上高度吻合, 这种空间异质性进一步证实水淹状况起到调节植被动态变化的作用。     

牛尾草中一新的对映-贝壳杉烷型二萜

从牛尾草[Isodon ternifolius(D.Don)Kudo]的地上部分分离得到一个新的对映－贝壳杉烷型二萜,命名为牛尾草素H（1）,通过波谱方法鉴定了它的结构。此外,还分离得到5个已知的对映－贝壳杉烷型二萜化合物：香茶菜醛（2）,长管香茶菜素A,E和G（3－5）,开展香茶菜素E（6）,以及木樨草素（7）,芹菜素（8）,α－香树脂醇（9）,乌索酸（10）和2α－羟基乌索酸（11）。