Heat maps for intramolecular communication in an RNP enzyme encoding glutamine

Allosteric signaling within large ribonucleoproteins modulates both catalytic function and biological specificity, but the spatial extent and quantitative magnitudes of long-distance free-energy couplings have yet to be well characterized. Here, we employ pre-steady-state kinetics to generate a comprehensive mapping of intramolecular communication in the glutaminyl-tRNA synthetase:tRNA(Gln) complex. Alanine substitution at 29 positions across the protein-RNA interface reveals distinct coupling amplitudes for glutamine binding and aminoacyl-tRNA formation on the enzyme, respectively, implying the existence of multiple signaling pathways. Structural models suggest that long-range signal propagation from the tRNA anticodon is dynamically driven, whereas shorter pathways are mediated by induced-fit rearrangements. Seven protein contacts with the distal tRNA vertical arm each weaken glutamine binding affinity across distances up to 40 ?, demonstrating that negative allosteric coupling plays a key role in enforcing the selective RNA-amino acid pairing at the heart of the genetic code.     

Epigenetic profiling of the antitumor natural product psammaplin A and its analogues

A collection of analogues of the dimeric natural product psammaplin A that differ in the substitution on the (halo)tyrosine aryl ring, the oxime and the diamine connection has been synthesized. The effects on cell cycle, induction of differentiation and apoptosis of the natural-product inspired series were measured on the human leukaemia U937 cell line. Epigenetic profiling included induction of p21(WAF1), effects on global H3 histone and tubulin acetylation levels as well as in vitro enzymatic assays using HDAC1, DNMT1, DNMT3A, SIRT1 and a peptide domain with p300/CBP HAT activity. Whereas the derivatives of psammaplin A with modifications in the length of the connecting chain, the oxime bond and the disulfide unit showed lower potency, the analogues with changes on the bromotyrosine ring exhibited activities comparable to those of the parent compound in the inhibition of HDAC1 and in the induction of apoptosis. The lack of HDAC1 activity of analogues modified on the disulfide bond suggests that its cleavage must occur in cells to produce the monomeric Zn(2+)-chelating thiol. This assumption is consistent with the molecular modelling of the complex of psammaplin A thiol with h-HDAC8. Only a weak inhibition of DNMT1, DNMT3A and residual activities with SIRT1 and a p300/CBP HAT peptide were measured for these compounds.     

Effect of different C3-aryl substituents on the antioxidant activity of 4-hydroxycoumarin derivatives

The antioxidant activity of 4-hydroxycoumarin synthetic derivatives and 4-methylumbelliferone were determined taking 4-hydroxycoumarin as the reference compound. Six 3-aryl-4-hydroxycoumarin derivatives were synthesized from 4-hydroxycoumarin as precursor in order to evaluate changes in their antioxidant properties due to C3-aryl substituent nature. Free radical scavenging capacities of these compounds against two different species DPPH(·) and ABTS(·+) and the protecting ability towards the β-carotene-linoleic acid co-oxidation enzymatically induced by lipoxygenase were measured. In addition, the relationship between the activities of these molecules against DPPH radical and the bond dissociation energy of O-H (BDE) calculated using methods of computational chemistry was evaluated.     

A specific protein disorder catalyzer of HIV-1 Nef

The HIV-1 auxiliary protein Nef is required for the onset and progression of AIDS in HIV-1-infected persons. Here, we have deciphered the mode of action of a second-generation inhibitor of Nef, DLC27-14, presenting a competitive IC(50) of ～16 μM measured by MALDI-TOF experiments. Thermal protein denaturation experiments revealed a negative effect on stability of Nef in the presence of a saturating concentration of the inhibitor. The destabilizing action of DLC27-14 was confirmed by a HIV protease-based experiment, in which the protease sensitivity of DLC27-14-bound Nef was three times as high as that of apo Nef. The only compatible docking modes of action for DLC27-14 suggest that DLC27-14 promotes an opening of two α-helices that would destabilize the Nef core domain. DLC27-14 thus acts as a specific protein disorder catalyzer that destabilizes the folded conformation of the protein. Our results open novel avenues toward the development of next-generation Nef inhibitors.     

Contemporary epidemiology of gout in the UK general population

Introduction  

The objective of this study was to investigate the contemporary incidence of gout, examine potential risk factors, and evaluate specific gout treatment patterns in the general population.     

Heterologous expression of manganese peroxidase in Aspergillus niger and its effect on phenanthrene removal from soil

A strain of Aspergillus niger, previously isolated from sugarcane bagasse because of its capacity to degrade phenanthrene in soil by solid culture, was used to express a manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium, aiming at increasing its polycyclic aromatic hydrocarbons degradation capacity. Transformants were selected based on their resistance to hygromycin B and the discoloration induced on Poly R-478 dye by the peroxidase activity. The recombinant A. niger SBC2-T3 strain developed MnP activity and was able to remove 95% of the initial phenanthrene (400 ppm) from a microcosm soil system after 17 days, whereas the wild strain removed 72% under the same conditions. Transformation success was confirmed by PCR amplification using gene-specific primers, and a single fragment (1,348 bp long, as expected) of the recombinant mnp1 was amplified in the DNA from transformants, which was absent from the parental strain.     

Differential N-glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice

Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.     

Near-infrared imaging method for the in vivo assessment of the biodistribution of nanoporous silicon particles

In the development of new nanoparticle-based technologies for therapeutic and diagnostic purposes, understanding the fate of nanoparticles in the body is crucial. We recently developed a multistage vector delivery system comprising biodegradable and biocompatible nanoporous silicon particles (first-stage microparticles [S1MPs]) able to host, protect, and deliver second-stage therapeutic and diagnostic nanoparticles (S2NPs) on intravenous injection. This delivery system aims at sequentially overcoming the biologic barriers en route to the target delivery site by separating and assigning tasks to the coordinated logic-embedded vectors constituting it. In this work, by conjugating a near-infrared dye on the surface of the S1MP without compromising the porous structure and potential loading of S2NPs, we were able to monitor the in vivo distribution of S1MPs in healthy mice using an optical imaging system. It was observed that particles predominantly accumulated in the liver and spleen at the end of 24 hours. Further quantification of S1MPs in the major organs of the animals by elemental analysis of silicon using inductively coupled plasma-atomic electron spectroscopy verified the accuracy of in vivo near-infrared imaging as a tool for evaluation of nanovector biodistribution.