Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α₁-antitrypsin (α₁AT) deficiency cases. It leads to increased polymerization of the serpin α₁AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α₁AT and analyzed their fluorescence properties. α₁AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α₁AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α₁AT compared to M α₁AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B.     

Transposase-transposase interactions in MOS1 complexes: a biochemical approach

Transposases are proteins that have assumed the mobility of class II transposable elements. In order to map the interfaces involved in transposase-transposase interactions, we have taken advantage of 12 transposase mutants that impair mariner transposase-transposase interactions taking place during transposition. Our data indicate that transposase-transposase interactions regulating Mos1 transposition are sophisticated and result from (i) active MOS1 dimerization through the first HTH of the N-terminal domain, which leads to inverted terminal repeat (ITR) binding; (ii) inactive dimerization carried by part of the C-terminal domain, which prevents ITR binding; and (iii) oligomerization. Inactive dimers are nonpermissive in organizing complexes that produce ITR binding, but the interfaces (or interactions) supplied in this state could play a role in the various rearrangements needed during transposition. Oligomerization is probably not due to a specific MOS1 domain, but rather the result of nonspecific interactions resulting from incorrect folding of the protein. Our data also suggest that the MOS1 catalytic domain is a main actor in the overall organization of MOS1, thus playing a role in MOS1 oligomerization. Finally, we propose that MOS1 behaves as predicted by the pre-equilibrium existing model, whereby proteins are found to exist simultaneously in populations with diverse conformations, monomers and active and inactive dimers for MOS1. We were able to identify several MOS1 mutants that modify this pre-existing equilibrium. According to their properties, some of these mutants will be useful tools to break down the remaining gaps in our understanding of mariner transposition.     

Changes over 46 years in plant community structure in a cleared brigalow (Acacia harpophylla) forest

Plant succession theory underpins the development of strategies for the conservation and regeneration of native communities. Current theory has been based largely on space‐for‐time rather than long‐term monitoring data, which have known limitations. There is general consensus that more site‐specific studies are needed to corroborate existing hypotheses. The target vegetation is a brigalow (Acacia harpophylla, Mimosaceae) forest in one of Australia's most endangered ecosystems, which was cleared and burnt in 1963. Forty quadrats were placed systematically within each of six 20 m × 20 m permanent plots. Presence, density and per cent canopy cover data were recorded for each species at 18 times over 46 years. Brigalow dominated the original vegetation, assumed dominance soon after clearing through massive root suckering and remained dominant throughout the study. It achieved maximum density within two years when severe intraspecific competition led to self‐thinning. After approximately 30 years, vacant niches appeared. Woody understorey species were slow to recolonise. Species richness and other diversity indices increased rapidly to a maximum after 2–4 years, declined until the 30th year when they again increased. This was the pattern of the species‐rich herbaceous layer; woody species showed a steady monotonic increase. The ‘hump‐shaped’ relationship between cover (biomass) and species richness was confirmed. This example fits the inhibition model for which few examples have been described. While the long‐term successional pattern is slightly confounded by climatic variability preceding sample surveys, this space‐for‐time study not only supports a bimodal pattern of diversity over time but also indicates that the relative species richness of the herbaceous and woody layers may explain the extreme variability reported in the literature.     

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis.Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04 Cmol_OA/Cmol_lac) and a higher P(3HB) production (18.88 g_P(3HB)/L) were achieved, comparing to the original recombinant strain (0.11 Cmol_OA/Cmol_lac and 7.8 g_P(3HB)/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28 Cmol_P(3HB)/Cmol_lac vs 0.10 Cmol_P(3HB)/Cmol_lac of the original strain).     

Delineation of key XRCC4/Ligase IV interfaces for targeted disruption of non‐homologous end joining DNA repair

Efficient DNA repair mechanisms frequently limit the effectiveness of chemotherapeutic agents that act through DNA damaging mechanisms. Consequently, proteins involved in DNA repair have increasingly become attractive targets of high‐throughput screening initiatives to identify modulators of these pathways. Disruption of the XRCC4‐Ligase IV interaction provides a novel means to efficiently halt repair of mammalian DNA double strand break repair; however; the extreme affinity of these proteins presents a major obstacle for drug discovery. A better understanding of the interaction surfaces is needed to provide a more specific target for inhibitor studies. To clearly define key interface(s) of Ligase IV necessary for interaction with XRCC4, we developed a competitive displacement assay using ESI‐MS/MS and determined the minimal inhibitory fragment of the XRCC4‐interacting region (XIR) capable of disrupting a complex of XRCC4/XIR. Disruption of a single helix (helix 2) within the helix‐loop‐helix clamp of Ligase IV was sufficient to displace XIR from a preformed complex. Dose‐dependent response curves for the disruption of the complex by either helix 2 or helix‐loop‐helix fragments revealed that potency of inhibition was greater for the larger helix‐loop‐helix peptide. Our results suggest a susceptibility to inhibition at the interface of helix 2 and future studies would benefit from targeting this surface of Ligase IV to identify modulators that disrupt its interaction with XRCC4. Furthermore, helix 1 and loop regions of the helix‐loop‐helix clamp provide secondary target surfaces to identify adjuvant compounds that could be used in combination to more efficiently inhibit XRCC4/Ligase IV complex formation and DNA repair. Proteins 2014; 82:187–194. © 2013 Wiley Periodicals, Inc.     

Phenol, 2,4-bis(1,1-dimethylethyl) of marine bacterial origin inhibits quorum sensing mediated biofilm formation in the uropathogen Serratia marcescens

Intercellular communication in bacteria (quorum sensing, QS) is an important phenomenon in disease dissemination and pathogenesis, which controls biofilm formation also. This study reports the anti-QS and anti-biofilm efficacy of seaweed Gracilaria gracilis associated Vibrio alginolyticus G16 against Serratia marcescens. Purification and mass spectrometric analysis revealed the active principle as phenol, 2,4-bis(1,1-dimethylethyl) [PD]. PD affected the QS regulated virulence factor production in S. marcescens and resulted in a significant (p < 0.05) reduction in biofilm (85%), protease (41.9%), haemolysin (69.9%), lipase (84.3%), prodigiosin (84.5%) and extracellular polysaccharide (84.62%) secretion without hampering growth, as evidenced by XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. qPCR analysis confirmed the down-regulation of the fimA, fimC, flhD and bsmA genes involved in biofilm formation. Apart from biofilm inhibition and disruption, PD increased the susceptibility of S. marcescens to gentamicin when administered synergistically, which opens another avenue for combinatorial therapy where PD can be used to enhance the efficacy of conventional antibiotics.