Umbelliferone aminoalkyl derivatives as inhibitors of human oxidosqualene-lanosterol cyclase

Human and murine lanosterol synthases (EC 5.4.99.7) were studied as targets of a series of umbelliferone aminoalkyl derivatives previously tested as inhibitors of oxidosqualene cyclases from other eukaryotes. Tests were carried out on cell cultures of human keratinocytes and mouse 3T3 fibroblasts incubated with radiolabeled acetate, and on homogenates prepared from yeast cells expressing human lanosterol synthase, incubated with radiolabeled oxidosqualene. In cell cultures of both human keratinocytes and mouse 3T3 fibroblasts, the observed inhibition of cholesterol biosynthesis was selective for oxidosqualene cyclase. The most active compounds bear an allylmethylamino chain in position-7 of the coumarin ring. The inhibition was critically dependent on the position and length of the inhibitor side chain, as well as on the type of aminoalkyl group inserted at the end of the same chain. Molecular docking analyses, carried out to clarify details of inhibitors/enzyme interactions, proved useful to explain the observed differences in inhibitory activities.     

The role of p38α mitogen-activated protein kinase gene in the HELLP syndrome

Mitogen-activated protein kinase (MAPK) p38α was shown to be implicated in the organogenesis of the placenta, and such placental alteration is crucial for the development of hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. We aimed to analyze for the first time human placental expression of MAPK p38α in pregnancies complicated by HELLP. The placental expression of MAPK p38α was investigated by semiquantitative polymerase chain reaction using cDNA extracted from placental tissue of 15 pregnancies with HELLP syndrome and 15 gestational age-matched controls. Seven patients with HELLP also had intrauterine fetal growth restriction (IUGR). In placenta from pregnancy complicated by HELLP, the expression of MAPK p38α is significantly decreased compared to the group with normal pregnancy (p < 0.001), while no difference was found between the HELLP and HELLP with IUGR subpopulations. Our study shows for the first time that MAPK p38α is expressed in the human placenta. Pregnancies with placental dysfunction and hypertensive complications are characterized by a significantly decreased expression of MAPK p38α. Our observations suggest that p38 MAPK signaling may be essential in placental angiogenesis and functioning.     

Effects of storage temperature on viability, germination and antioxidant metabolism in Ginkgo biloba L. seeds.

The behaviour of the Ginkgo biloba L. seeds was studied during storage at 4 and 25 degrees C. When stored at 25 degrees C, all the seeds died in 6 months. Cold temperatures preserved seed tissue viability for 1 year but did not preserve their capability to germinate, since such capability decreased after 6 months. A significant increase in lipid peroxidation occurred in the seed both in the embryo and in the endosperm. During storage a progressive deterioration of the endosperm tissues was evident. The two major water soluble antioxidants, ascorbate (ASC) and glutathione (GSH), showed different behaviour in the two conditions of storage and in the two main structures of the seed, the embryo and the endosperm. The ASC content of embryos and endosperms remained quite unchanged in the first 9 months at 4 degrees C, then increased. At 25 degrees C a significant decrease in the ASC content in the embryos was evident, whereas it remained more stable in the endosperm. The GSH pool decreased at both storage temperatures in the embryos. As far as the ASC-GSH redox enzymes are concerned, their activities decreased with storage, but changes appeared to be time-dependent more than temperature-dependent, with the exception of the endosperm ascorbate free radical (AFR) reductase (EC 1.6.5.4), the activity of which rapidly decreased at 25 degrees C. Therefore overall the antioxidant enzymes were scarcely regulated and unable to counteract oxidative stress occurring during the long-term storage.     

Thyroid hormone regulation of cell migration and oxidative metabolism in polymorphonuclear leukocytes: clinical evidence in thyroidectomized subjects on thyroxine replacement therapy

Migration and superoxide anion (O2-) generation were studied in polymorphonuclear leukocytes (PMNs) from 14 athyreotic patients, previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice: in hypothyroidism (visit 1) and after TSH-suppressive L-T4 replacement therapy (visit 2). Random migration and N-formyl-Met-Leu-Phe (fMLP) 0.1-microM induced chemotaxis were similar in cells from patients at both visit 1 and visit 2 and from healthy controls. On the contrary, resting O2- generation in cells from patients was significantly lower than control values, both at visit 1 and 2. At visit 1, fMLP 0.1 muM-induced O2- generation was significantly lower than control values, while phorbol-myristate acetate (PMA) 100-ng/ml induced O2- generation was similar in cells from patients and from controls. At visit 2 both responses increased, resulting in fMLP-induced O2- generation superimposable to control values and PMA-induced O2- generation significantly higher with respect to both visit 1 and cells from controls. In vitro exposure of PMNs from healthy subjects to L-T4 did not affect O2- generation in resting cells, and significantly increased that induced by fMLP or PMA only at high, supra-physiological concentrations. Neither TSH nor T3 had significant effects at any of the concentrations tested. The present results document the existence of a correlation between thyroid status and oxidative metabolism of human PMNs, which is however unlikely to depend upon a direct action of thyroid hormones on these cells.     

Rationalisation of the Differences between APOBEC3G Structures from Crystallography and NMR Studies by Molecular Dynamics Simulations

The human APOBEC3G (A3G) protein is a cellular polynucleotide cytidine deaminase that acts as a host restriction factor of retroviruses, including HIV-1 and various transposable elements. Recently, three NMR and two crystal structures of the catalytic deaminase domain of A3G have been reported, but these are in disagreement over the conformation of a terminal β-strand, β2, as well as the identification of a putative DNA binding site. We here report molecular dynamics simulations with all of the solved A3G catalytic domain structures, taking into account solubility enhancing mutations that were introduced during derivation of three out of the five structures. In the course of these simulations, we observed a general trend towards increased definition of the β2 strand for those structures that have a distorted starting conformation of β2. Solvent density maps around the protein as calculated from MD simulations indicated that this distortion is dependent on preferential hydration of residues within the β2 strand. We also demonstrate that the identification of a pre-defined DNA binding site is prevented by the inherent flexibility of loops that determine access to the deaminase catalytic core. We discuss the implications of our analyses for the as yet unresolved structure of the full-length A3G protein and its biological functions with regard to hypermutation of DNA.     

On the Potential Contribution of Microfungi to the Decomposition of Reed Leaf Detritus in a Coastal Lagoon: A Laboratory and Field Experiment

The potential contribution of microfungi to reed decomposition in a coastal habitat (Le Cesine Lagoon, Italy) was investigated under laboratory and field conditions. Leaf pack mass and surface loss, ergosterol content and O₂ uptake were used to construct carbon budgets and an empirical ergosterol‐to‐O₂ uptake relationship based on literature data was used to estimate the contribution of microfungi. Under laboratory conditions, reed carbon loss was entirely due to leaching and microbial respiration. In contrast, C losses observed in the field were accounted for by microbial respiration and macroinvertebrate shredding almost equally. Microfungi were estimated to account for 98% and 69% of microbially‐respired carbon under laboratory and field conditions, respectively. Our results provide a preliminary, quantitative assessment of microfungal contribution to reed decomposition in brackish habitats. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Chitosan molecular structure as a function of N-acetylation

Molecular dynamics simulations have been carried out to characterize the structure and solubility of chitosan nanoparticle‐like structures as a function of the deacetylation level (0, 40, 60, and 100%) and the spatial distribution of the N‐acetyl groups in the particles. The polysaccharide chains of highly N‐deacetylated particles where the N‐acetyl groups are uniformly distributed present a high flexibility and preference for the relaxed two‐fold helix and five‐fold helix motifs. When these groups are confined to a given region of the particle, the chains adopt preferentially a two‐fold helix with ? and ψ values close to crystalline chitin. Nanoparticles with up to 40% acetylation are moderately soluble, forming stable aggregates when the N‐acetyl groups are unevenly distributed. Systems with 60% or higher N‐acetylation levels are insoluble and present similar degrees of swelling regardless the distribution of their N‐acetyl groups. Overall particle solvation is highly affected by electrostatic forces resulting from the degree of acetylation. The water mobility and orientation around the polysaccharide chains affects the stability of the intramolecular O3‐HO3_(n)···O5_{(n +1)} hydrogen bond, which in turn controls particle aggregation. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 448–460, 2011.     

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.     

EBV lytic-phase protein BGLF5 contributes to TLR9 downregulation during productive infection

Viruses use a wide range of strategies to modulate the host immune response. The human gammaherpesvirus EBV, causative agent of infectious mononucleosis and several malignant tumors, encodes proteins that subvert immune responses, notably those mediated by T cells. Less is known about EBV interference with innate immunity, more specifically at the level of TLR-mediated pathogen recognition. The viral dsDNA sensor TLR9 is expressed on B cells, a natural target of EBV infection. Here, we show that EBV particles trigger innate immune signaling pathways through TLR9. Furthermore, using an in vitro system for productive EBV infection, it has now been possible to compare the expression of TLRs by EBV(-) and EBV(+) human B cells during the latent and lytic phases of infection. Several TLRs were found to be differentially expressed either in latently EBV-infected cells or after induction of the lytic cycle. In particular, TLR9 expression was profoundly decreased at both the RNA and protein levels during productive EBV infection. We identified the EBV lytic-phase protein BGLF5 as a protein that contributes to downregulating TLR9 levels through RNA degradation. Reducing the levels of a pattern-recognition receptor capable of sensing the presence of EBV provides a mechanism by which the virus could obstruct host innate antiviral responses.