Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

Molecular phylogeny of the fungus gnat subfamilies Gnoristinae and Mycomyinae,and their position within Mycetophilidae (Diptera)

The phylogeny of the fungus gnat family Mycetophilidae (Diptera) is reconstructed with a focus on the species‐rich and taxonomically difficult subfamilies Gnoristinae and Mycomyinae. The multigene phylogenetic analyses are based on five nuclear (18S, 28S, CAD, MCS, ITS2) and four mitochondrial (12S, 16S, COI, CytB) gene markers. The analyses strongly support the monophyly of Mycetophilidae and the subfamilies Manotinae, Sciophilinae, Leiinae, and Mycomyinae, although Gnoristinae is paraphyletic with respect to Mycetophilinae. All the genera and groups of genera included are supported as monophyletic, except for Acomoptera Vockeroth, Boletina Staeger, Dziedzickia Johannsen, Ectrepesthoneura Enderlein, and Neoempheria Osten Sacken. Ancestral character state reconstructions were applied to two morphological features present in Gnoristinae and Mycomyinae (i.e. presence of setae on wing membrane and wing vein R₄) in order to assess their evolution. The wing vein R₄ appears as an unstable character, spread throughout different clades. A dated phylogeny of the family Mycetophilidae showed that most of the subfamilies of Mycetophilidae originated and diversified during the Cretaceous. The youngest subfamilies, originated in the Paleogene, appear to be Mycomyinae and Mycetophilinae.     

Further results on the survival of a gene represented in a founder population

This paper is concerned with gene survival in a population which may increase without density dependence according to a generalization of the Moran model for haploid individuals. A selective advantage to one allele and the possibility of differential reproductive rates are allowed. Simple conditions are given for ultimate homozygosity to be certain and for the possibility of ultimate polymorphism. The results complement and extend those of Heyde (1981, 1982).     

Computer Simulation of Assembly and Co-operativity of Hexameric AAA ATPases

AAA ATPases form a functionally diverse superfamily of proteins. Most members form homo-hexameric ring complexes, are catalytically active only in the fully assembled state, and show co-operativity among the six subunits. The mutual dependence among the subunits is clearly evidenced by the fact that incorporation of mutated, inactive subunits can decrease the activity of the remaining wild type subunits. For the first time, we develop here models to describe this form of allostery, evaluate them in a simulation study, and test them on experimental data. We show that it is important to consider the assembly reactions in the kinetic model, and to define a formal inhibition scheme. We simulate three inhibition scenarios explicitly, and demonstrate that they result in differing outcomes. Finally, we deduce fitting formulas, and test them on real and simulated data. A non-competitive inhibition formula fitted experimental and simulated data best. To our knowledge, our study is the first one that derives and tests formal allosteric schemes to explain the inhibitory effects of mutant subunits on oligomeric enzymes.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.