Heterochromatin loss as a determinant of progerin‐induced DNA damage in Hutchinson–Gilford Progeria

Hutchinson–Gilford progeria is a premature aging syndrome caused by a truncated form of lamin A called progerin. Progerin expression results in a variety of cellular defects including heterochromatin loss, DNA damage, impaired proliferation and premature senescence. It remains unclear how these different progerin‐induced phenotypes are temporally and mechanistically linked. To address these questions, we use a doxycycline‐inducible system to restrict progerin expression to different stages of the cell cycle. We find that progerin expression leads to rapid and widespread loss of heterochromatin in G1‐arrested cells, without causing DNA damage. In contrast, progerin triggers DNA damage exclusively during late stages of DNA replication, when heterochromatin is normally replicated, and preferentially in cells that have lost heterochromatin. Importantly, removal of progerin from G1‐arrested cells restores heterochromatin levels and results in no permanent proliferative impediment. Taken together, these results delineate the chain of events that starts with progerin expression and ultimately results in premature senescence. Moreover, they provide a proof of principle that removal of progerin from quiescent cells restores heterochromatin levels and their proliferative capacity to normal levels.     

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     

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.     

TWEAK as a target for therapy in systemic lupus erythematosus

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a recently identified proinflammatory cytokine of the TNF superfamily. Through activation of the fibroblast growth factor-inducible 14 (Fn14) receptor, TWEAK regulates cell proliferation, cell death and inflammation. The available evidences have indicated that TWEAK might be a target for therapeutic intervention in renal, vascular injury and neuropathy. Since renal, vascular and neuropsychiatric complications are frequently encountered in systemic lupus erythematosus (SLE)—a systemic autoimmune disease, TWEAK-Fn14 pathway may be implicated in the pathogenesis of SLE. In this review, we will discuss the TWEAK-Fn14 pathway and the therapeutic potential of modulating this pathway in SLE.     

Phenolic composition and antioxidant properties of some traditionally used medicinal plants affected by the extraction time and hydrolysis

Introduction – Polyphenolic phytochemicals in traditionally used medicinal plants act as powerful antioxidants, which aroused an increasing interest in their application in functional food development. Objective – The effect of extraction time (5 and 15 min) and hydrolysis on the qualitative and quantitative content of phenolic compounds and antioxidant capacity of six traditionally used medicinal plants (Melissa officinalis L., Thymus serpyllum L., Lavandula officinalis Miller, Rubus fruticosus L., Urtica dioica L., and Olea europea L.) were investigated. Methodology – The content of total phenols, flavonoids, flavan‐3‐ols and tannins was determined using UV/Vis spectrophotometric methods, while individual phenolic acids, flavones and flavonols were separated and detected using HPLC analysis. Also, to obtain relevant data on the antioxidant capacity, two different assays, (2,2‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid) (ABTS) radical scavenging and ferric reducing/antioxidant power (FRAP) assays were used. Results – The extraction efficiency of phenolics, as well as the antioxidant capacity of plant extracts, was affected by both prolonged extraction and hydrolysis. The overall highest content of phenolic compounds was determined in hydrolyzed extract of blackberry leaves (2160 mg GAE/L), followed by the non‐hydrolyzed extract of lemon balm obtained after 15 min of extraction (929.33 mg GAE/L). The above extracts also exhibited the highest antioxidant capacity, while extracts of olive leaves were characterized with the lowest content of phenolic compounds, as well as the lowest antioxidant capacity. The highest content of rosmarinic acid, as the most abundant phenolic compound, was determined in non‐hydrolyzed extract of lemon balm, obtained after 15 min of extraction. Although the hydrolysis provided the highest content of polyphenolic compounds, longer extraction time (15 min) was more efficient to extract these bioactives than shorter extraction duration (5 min). Conclusion – The distribution of detected phenolic compounds showed a wide variability with regard to their botanical origin. Examined medicinal plants showed to be a valuable supplement to a daily intake of bioactive compounds. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

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。     

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.     

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.