Chemical synthesis of 4-azido-β-galactosamine derivatives for inhibitors of <Emphasis Type="Italic">N</Emphasis>-acetylgalactosamine 4-sulfate 6-<Emphasis Type="Italic">O</Emphasis>-sulfotransferase

Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-β-GalNAc derivatives and 4-azido-β-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-β-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC₅₀ value ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.     

A new robust kinetic assay for DAP epimerase activity

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase–DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP⁺ to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.     

Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

Diaminopimelate (DAP) epimerase is involved in the biosynthesis of meso-DAP and lysine, which are important precursors for the synthesis of peptidoglycan, housekeeping proteins, and virulence factors in bacteria. Accordingly, DAP epimerase is a promising antimicrobial target. Previous studies report that DAP epimerase exists as a monomeric enzyme. However, we show using analytical ultracentrifugation, X-ray crystallography, and enzyme kinetic analyses that DAP epimerase from Escherichia coli exists as a functional dimer in solution and the crystal state. Furthermore, the 2.0-Å X-ray crystal structure of the E. coli DAP epimerase dimer shows for the first time that the enzyme exists in an open, active conformation. The importance of dimerization was subsequently probed by using site-directed mutagenesis to generate a monomeric mutant (Y268A). Our studies show that Y268A is catalytically inactive, thus demonstrating that dimerization of DAP epimerase is essential for catalysis. Molecular dynamics simulations indicate that the DAP epimerase monomer is inherently more flexible than the dimer, suggesting that dimerization optimizes protein dynamics to support function. Our findings offer insight into the development of novel antimicrobial agents targeting the dimeric antibiotic target DAP epimerase.     

The species richness of click beetles in ancient pasture woodland benefits from a high level of sun exposure

Forests support high concentrations of species and beetles in particular are often used to evaluate forest biodiversity. Ancient pasture woodlands are facing a major decline in Europe mainly due to the abandonment of traditional management and subsequent succession. We studied click beetles (Coleoptera: Elateridae) in one of the largest central-European remnants of pasture woodland in Lány Game Park (Czech Republic) using flight interception traps placed at standing veteran trees. The gradient of sun-exposure, circumference of stem, height and vitality of tree and tree species were studied in relation to the species richness of click beetles and their ecological groups. Total species richness reached nearly one half of the recently documented fauna in the study area and species accumulations showed us that the majority of species were represented. Most species preferred solitary trees in sun-exposed habitats and avoided shaded trees in closed canopies. The same results were obtained for ecological groups, such as saproxylic and non-saproxylic species, functional groups and guilds. Our results showed that the species richness of one of the most ecologically diverse beetle families, click beetles, benefits from a high level of sun exposure. Thus, the long spatial and temporal continuity of sun-exposed veteran trees could be a good predictor for sustainable forest management.     

Both human ferredoxins equally efficiently rescue ferredoxin deficiency in Trypanosoma brucei

Ferredoxins are highly conserved proteins that function universally as electron transporters. They not only require Fe‐S clusters for their own activity, but are also involved in Fe‐S formation itself. We identified two homologues of ferredoxin in the genome of the parasitic protist Trypanosoma brucei and named them TbFdxA and TbFdxB. TbFdxA protein, which is homologous to other eukaryotic mitochondrial ferredoxins, is essential in both the procyclic (= insect‐transmitted) and bloodstream (mammalian) stage, but is more abundant in the active mitochondrion of the former stage. Depletion of TbFdxA caused disruption of Fe‐S cluster biogenesis and lowered the level of intracellular haem. However, TbFdxB, which is present exclusively within kinetoplastid flagellates, was non‐essential for the procyclic stage, and double knock‐down with TbFdxA showed this was not due to functional redundancy between the two homologues. Heterologous expressions of human orthologues HsFdx1 and HsFdx2 fully rescued the growth and Fe‐S‐dependent enzymatic activities of TbFdxA knock‐down. In both cases, the genuine human import signals allowed efficient import into the T. brucei mitochondrion. Given the huge evolutionary distance between trypanosomes and humans, ferredoxins clearly have ancestral and highly conserved function in eukaryotes and both human orthologues have retained the capacity to participate in Fe‐S cluster assembly.     

过量表达水杨酸羟基酶NahG降低烟草对青枯菌的抗性

研究构建了pMDC32-NahG植物表达载体,并获得了其超表达的转基因烟草T1代株系。结果表明:这些株系与其野生型植株没有明显表型差异,但却表现出较野生型植株更敏感的青枯菌侵染抗性。同时,研究还发现NahG的超表达还显著提高了茉莉酸(JA)-依赖的基因NtPR1-b的表达,降低了(SA)-依赖的相关基因NtPR3和NtPRQ的表达。这表明SA累积的缺陷降低了烟草对青枯菌侵染的抗性。     

Fabrication and characterization of tosyl‐activated magnetic and nonmagnetic monodisperse microspheres for use in microfluic‐based ferritin immunoassay

This article describes the preparation of tosyl‐activated nonmagnetic poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) [P(HEMA‐GMA)] microspheres by dispersion polymerization and tosyl‐activated magnetic poly(2‐hydroxyethyl methacrylate‐co‐ethylene dimethacrylate) [P(HEMA‐EDMA)] microspheres by multistep swelling polymerization method and precipitation of iron oxide inside the pores. These new approaches show that monodisperse microspheres, 2.3 µm, respectively 4.1 µm, in diameter can be produced in high yields avoiding aggregation and with the advantage of being free of aromatic moieties. To demonstrate their potential for diagnostic applications, both types of microparticles have been coated with capture and detection antibodies (DAs), respectively. Immunoassay protocols have then been developed for the dosage of ferritin using an automated affinity platform combining microchannel chips and electrochemical detection. The assay performance using the above magnetic microspheres has been compared with that obtained with commercial tosyl‐activated beads. Finally, the possibility to combine functionalized magnetic and nonmagnetic microspheres has been evaluated in view of amplifying the number of enzymatic labels in the immuno‐complex. At a ferritin concentration of 119.6 ng/mL, a signal‐to‐noise ratio of 150.5 is obtained using 0.2 mg/mL of anti‐ferritin‐coated P(HEMA‐GMA)‐DA microspheres against a value of 158.8 using free DA in solution. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 532–542, 2013     

中国遗传学会科普工作会议在京召开

培养温度是花药培养的一个十分重要的条 件。但是有关这方面研究的报道是不多的。特 别是早期的一些报道,不但内容比较简单,而且 试验的温度范围都在28℃ 以下^[7-9.14.16]近年 来甘肃农科院等一些单位开始试验用高温培 养,得到良好的效果^[4.5.10.15不过陈英等在水 稻上初步看到在高温培养下花粉愈伤组织的诱 导率虽然提高,但愈伤组织的分化能力有降低 的趋势,特别是当愈伤组织转分化培养基的时 间偏晚时更是如此^[5]。我们过去在小麦上也见 到过类似现象^[1]。因此近年来我们探索了在高 温下诱导的小麦花粉愈伤组织的分化能力的保 持间题。但在这一研究中又看到不同基因型对 培养温度有不同的反应,从而又就这种对培养 温度反应的基因型差异做了初步的遗传学分 析。本文先报道关于基因型差异方面的结果。 其他结果将另文报道。     

Habitat use,but not gene flow,is influenced by human activities in two ecotypes of Egyptian fruit bat (Rousettus aegyptiacus)

Understanding the ecological, behavioural and evolutionary response of organisms to changing environments is of primary importance in a human‐altered world. It is crucial to elucidate how human activities alter gene flow and what are the consequences for the genetic structure of a species. We studied two lineages of the Egyptian fruit bat (Rousettus aegyptiacus) throughout the contact zone between mesic and arid Ecozones in the Middle East to evaluate the species' response to the growing proportion of human‐altered habitats in the desert. We integrated population genetics, morphometrics and movement ecology to analyse population structure, morphological variation and habitat use from GPS‐ or radio‐tagged individuals from both desert and Mediterranean areas. We classified the spatial distribution and environmental stratification by describing physical–geographical conditions and land cover. We analysed this information to estimate patch occupancy and used an isolation‐by‐resistance approach to model gene flow patterns. Our results suggest that lineages from desert and Mediterranean habitats, despite their admixture, are isolated by environment and by adaptation supporting their classification as ecotypes. We found a positive effect of human‐altered habitats on patch occupancy and habitat use of fruit bats by increasing the availability of roosting and foraging areas. While this commensalism promotes the distribution of fruit bats throughout the Middle East, gene flow between colonies has not been altered by human activities. This discrepancy between habitat use and gene flow patterns may, therefore, be explained by the breeding system of the species and modifications of natal dispersal patterns.