Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).     

A model for the architecture of the hemocyanin from the arthropod Squilla mantis (Crustacea, Stomatopoda)

Squilla mantis hemocyanin is composed of two hexameric subunits but has electron microscopic profiles different from other bis-hexameric hemocyanins, e.g. Astacus and Homarus. We distinguished three different electron microscopic profiles of S. mantis hemocyanin: two sideviews and a topview. These profiles were studied using computer image alignment and correspondence analysis [Van Heel, M. and Frank, J. (1981) Ultramicroscopy 6, 187 - 194]. With the results of this analysis we were able to build a three-dimensional model for the quaternary structure of this hemocyanin. In this model the two hexamers are stacked in such a way that their hexagonal surfaces overlap to about 60% of their width. In the overlap area four subunits are arranged in two different interhexameric pairs, each forming a bridging area between the two hexamers.     

Disrupted phylogeographical microsatellite and chloroplast DNA patterns indicate a vicariance rather than long‐distance dispersal origin for the disjunct distribution of the Chilean endemic Dioscorea biloba (Dioscoreaceae) around the Atacama Desert

Aim The Chilean endemic Dioscorea biloba (Dioscoreaceae) is a dioecious geophyte that shows a remarkable 600 km north–south disjunction in the peripheral arid area of the Atacama Desert. Its restricted present‐day distribution and probable Neogene origin indicate that its populations have a history linked to that of the Atacama Desert, making this an ideal model species with which to investigate the biogeography of the region. Location Chile, Atacama Desert and peripheral arid area. Methods Two hundred and seventy‐five individuals from nine populations were genotyped for seven nuclear microsatellite loci, and plastid trnL–F and trnT–L sequences were obtained for a representative subset of these. Analyses included the estimation of genetic diversity and population structure through clustering, Bayesian and analysis of molecular variance analyses, and statistical parsimony networks of chloroplast haplotypes. Isolation by distance was tested against alternative dispersal hypotheses. Results Microsatellite markers revealed moderate to high levels of genetic diversity within populations, with those from the southern Limarí Valley showing the highest values and northern populations showing less exclusive alleles. Bayesian analysis of microsatellite data identified three genetic groups that corresponded to geographical ranges. Chloroplast phylogeography revealed no haplotypes shared between northern and southern ranges, and little haplotype sharing between the two neighbouring southern valleys. Dispersal models suggested the presence of extinct hypothetical populations between the southern and northern ranges. Main conclusions Our results are consistent with prolonged isolation of the northern and southern groups, mediated by the life‐history traits of the species. Significant isolation was revealed at both large and moderate distances as gene flow was not evident even between neighbouring valleys. Bayesian analyses of microsatellite and chloroplast haplotype diversity identified the southern area of Limarí as the probable area of origin of the species. Our data do not support recent dispersal of D. biloba from the southern range into Antofagasta, but indicate the fragmentation of an earlier wider range, concomitant with the Pliocene–Pleistocene climatic oscillations, with subsequent extinctions of the Atacama Desert populations and the divergence of the peripheral ones as a consequence of genetic drift.     

DFT studies of the phenol adsorption on boron nitride sheets

We perform first principles total energy calculations to investigate the atomic structures of the adsorption of phenol (C₆H₅OH) on hexagonal boron nitride (BN) sheets. Calculations are done within the density functional theory as implemented in the DMOL code. Electron-ion interactions are modeled according to the local-spin-density-approximation (LSDA) method with the Perdew-Wang parametrization. Our studies take into account the hexagonal h-BN sheets and the modified by defects d-BN sheets. The d-BN sheets are composed of one hexagon, three pentagons and three heptagons. Five different atomic structures are investigated: parallel to the sheet, perpendicular to the sheet at the B site, perpendicular to the sheet at the N site, perpendicular to the central hexagon and perpendicular to the B-N bond (bridge site). To determine the structural stability we apply the criteria of minimum energy and vibration frequency. After the structural relaxation phenol molecules adsorb on both h-BN and d-BN sheets. Results of the binding energies indicate that phenol is chemisorbed. The polarity of the system increases as a consequence of the defects presence which induces transformation from an ionic to covalent bonding. The elastic properties on the BN structure present similar behavior to those reported in the literature for graphene.     

Development of species-specific primers for rapid identification by PCR of the ecologically important pathogen Fusarium keratoplasticum from isolated and environmental samples

The genus Fusarium contains many fungal species known to be pathogenic to animals and plants alike. One species complex within this genus, the Fusarium solani species complex (FSSC), is of particular concern due to its high numbers of pathogenic members. FSSC members are known to contribute significantly to plant, human and other animal fungal disease. One member of the FSSC, Fusarium keratoplasticum, is of particular ecological concern and has been implicated in low hatching success of endangered sea turtle eggs, as well as contribute to human and other animal Fusarium pathogenesis. Species-specific primers for molecular identification of F. keratoplasticum currently do not exist to our knowledge, making rapid identification, tracking and quantitation of this pathogenic fungus difficult. The objective of this study was to develop primers specific to F. keratoplasticum that could be applied to DNA from isolated cultures as well as total (mixed) DNA from environmental samples. RPB2 sequence from 109 Fusarium isolates was aligned and analysed to determine nucleotide polymorphisms specific to F. keratoplasticum useful for primer design. A set of primers were generated and found to be effective for identification of F. keratoplasticum from total DNA extracted from sand surrounding sea turtle nesting sites.     

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.