Expression and physicochemical characterization of an extracellular segment of the receptor protein tyrosine phosphatase IA-2

The receptor protein tyrosine phosphatase superfamily (RPTP) includes proteins with a single transmembrane, one or more intracellular phosphatase, and a variety of extracellular domains. The 106-kDa insulinoma-associated protein (IA-2, ICA512) receptor is unique among RPTP members because: (a) it has a single, phosphatase-like intracellular domain identified as one of the most prominent self antigens in autoimmune diabetes; (b) its extracellular region bears no sequence similarity to known domains; (c) it is present in the membrane of secretory granules in neurons and pancreatic beta-cells where it suffers a complex processing; and (d) it has very poorly understood biological properties. In this work, we describe the expression, purification, and physicochemical characterization of residues 449-576 of IA-2 (IA-2ec(449-576)). Judging from CD, fluorescence, hydrodynamic, and thermal unfolding analyses, this fragment forms an autonomously folding unit with tight packing and well-defined secondary and tertiary structure. CD analysis suggests that about 25% of IA-2ec(449-576) residues are alpha-helical, whereas about the same amount are in beta-sheet structure. The availability of soluble and folded IA-2ec(449-576) is a step forward toward the characterization of a part of IA-2 at atomic detail, which may provide new insight in the biology of diabetes, the neurotransmission process, and the dynamic of secretory granules.     

Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 Sodium channels

The three-dimensional structures of the long-chain mammalian scorpion β-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed α/β fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na(v) channels.     

New tricks of an old pattern: structural versatility of scorpion toxins with common cysteine spacing

Scorpion venoms are a rich source of K(+) channel-blocking peptides. For the most part, they are structurally related small disulfide-rich proteins containing a conserved pattern of six cysteines that is assumed to dictate their common three-dimensional folding. In the conventional pattern, two disulfide bridges connect an α-helical segment to the C-terminal strand of a double- or triple-stranded β-sheet, conforming a cystine-stabilized α/β scaffold (CSα/β). Here we show that two K(+) channel-blocking peptides from Tityus scorpions conserve the cysteine spacing of common scorpion venom peptides but display an unconventional disulfide pattern, accompanied by a complete rearrangement of the secondary structure topology into a CS helix-loop-helix fold. Sequence and structural comparisons of the peptides adopting this novel fold suggest that it would be a new elaboration of the widespread CSα/β scaffold, thus revealing an unexpected structural versatility of these small disulfide-rich proteins. Acknowledgment of such versatility is important to understand how venom structural complexity emerged on a limited number of molecular scaffolds.     

Linkers of Cell Polarity and Cell Cycle Regulation in the Fission Yeast Protein Interaction Network

The study of gene and protein interaction networks has improved our understanding of the multiple, systemic levels of regulation found in eukaryotic and prokaryotic organisms. Here we carry out a large-scale analysis of the protein-protein interaction (PPI) network of fission yeast (Schizosaccharomyces pombe) and establish a method to identify ‘linker’ proteins that bridge diverse cellular processes - integrating Gene Ontology and PPI data with network theory measures. We test the method on a highly characterized subset of the genome consisting of proteins controlling the cell cycle, cell polarity and cytokinesis and identify proteins likely to play a key role in controlling the temporal changes in the localization of the polarity machinery. Experimental inspection of one such factor, the polarity-regulating RNB protein Sts5, confirms the prediction that it has a cell cycle dependent regulation. Detailed bibliographic inspection of other predicted ‘linkers’ also confirms the predictive power of the method. As the method is robust to network perturbations and can successfully predict linker proteins, it provides a powerful tool to study the interplay between different cellular processes.     

SPARC Controls Melanoma Cell Plasticity through Rac1

Cell transition to a more aggressive mesenchymal-like phenotype is a hallmark of cancer progression that involves different steps and requires tightly regulated cell plasticity. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a matricellular protein that promotes this transition in various malignant cell types, including melanoma cells. We found that suppression of SPARC expression in human melanoma cells compromised cell migration, adhesion, cytoskeleton structure, and cell size. These changes involved the Akt/mTOR pathway. Re-expression of SPARC or protein addition restored all the cell features. Suppression of SPARC expression was associated with increased Rac1-GTP levels and its membrane localization. Expression of the dominant negative mutant of Rac1 counteracted almost all the changes observed in SPARC-deficient cells. Overall, these data suggest that most of the SPARC-mediated effects occurred mainly through the blockade of Rac1 activity.     

Nutritional symbionts of a putative vector, <Emphasis Type="Italic">Xyleborus bispinatus</Emphasis>, of the laurel wilt pathogen of avocado, <Emphasis Type="Italic">Raffaelea lauricola</Emphasis>

Ambrosia beetles subsist on fungal symbionts that they carry to, and cultivate in, their natal galleries. These symbionts are usually saprobes, but some are phytopathogens. Very few ambrosial symbioses have been studied closely, and little is known about roles that phytopathogenic symbionts play in the life cycles of these beetles. One of the latter symbionts, Raffaelea lauricola, causes laurel wilt of avocado, Persea americana, but its original ambrosia beetle partner, Xyleborus glabratus, plays an uncertain role in this pathosystem. We examined the response of a putative, alternative vector of R. lauricola, Xyleborus bispinatus, to artificial diets of R. lauricola and other ambrosia fungi. Newly eclosed, unfertilized females of X. bispinatus were reared in no-choice assays on one of five different symbionts or no symbiont. Xyleborus bispinatus developed successfully on R. lauricola, R. arxii, R. subalba and R. subfusca, all of which had been previously recovered from field-collected females of X. bispinatus. However, no development was observed in the absence of a symbiont or on another symbiont, Ambrosiella roeperi, recovered from another ambrosia beetle, Xylosandrus crassiusculus. In the no-choice assays, mycangia of foundress females of X. bispinatus harbored significant colony-forming units of, and natal galleries that they produced were colonized with, the respective Raffaelea symbionts; with each of these fungi, reproduction, fecundity and survival of the beetle were positively impacted. However, no fungus was recovered from, and reproduction did not occur on, the A. roeperi and no symbiont diets. These results highlight the flexible nature of the ambrosial symbiosis, which for X. bispinatus includes a fungus with which it has no evolutionary history. Although the “primary” symbiont of the neotropical X. bispinatus is unclear, it is not the Asian R. lauricola.     

Enhanced production of scopolin bySolanum aviculare cells immobilised within Ca-alginate gel beads

Summary Different matrices, obtained by varying calcium (0.1 to 1.5M) and alginate (1 to 1.5%) concentrations, were used to study the influence of immobilisation parameters on the behaviour ofS. aviculare. A significant modulation of cell growth, cell release, and scopolin production and excretion has been observed. Physiological and morphological characteristics ofSolanum aviculare cells immobilised within Ca-alginate beads were notably different from those of suspended cells. ImmobilisedS. aviculare have accumulated scopolin (up to 120 g·g^–1 FWB) within beads and excreted it into the culture medium (up to 8 g·g^–1 FWB). Contrary to suspended cells which have accumulated only traces of this metabolite within intracellular compartments (1 g·g^–1 FWB), no scopolin has been found into the culture medium.Abbreviations ANA -Naphthaleneacetic acid - HPLC high performance liquid chromatography - FWB fresh weight biomass - LS medium Linsmaier and Skoog medium - MS mass spectroscopy - NMR nuclear magnetic resonance - r² coefficient of determination - s standard deviation     

Dependence of lung injury on surface tension during low-volume ventilation in normal open-chest rabbits.

To evaluate the role of pulmonary surfactant in the prevention of lung injury caused by mechanical ventilation (MV) at low end-expiratory volumes, lung mechanics and morphometry were assessed in three groups of eight normal, open-chest rabbits ventilated for 3-4 h at zero end-expiratory pressure (ZEEP) with physiological tidal volumes (Vt = 10 ml/kg). One group was left untreated (group A); the other two received surfactant intratracheally (group B) or aerosolized dioctylsodiumsulfosuccinate (group C) before MV on ZEEP. Relative to initial MV on positive end-expiratory pressure (PEEP; 2.3 cmH(2)O), quasi-static elastance (Est) and airway (Rint) and viscoelastic resistance (Rvisc) increased on ZEEP in all groups. After restoration of PEEP, only Rint (124%) remained elevated in group A, only Est (36%) was significantly increased in group B, whereas in group C, Est, Rint, and Rvisc were all markedly augmented (274, 253, and 343%). In contrast, prolonged MV on PEEP had no effect on lung mechanics of eight open-chest rabbits (group D). Lung edema developed in group C (wet-to-dry ratio = 7.1), but not in the other groups. Relative to group D, both groups A and C, but not B, showed histological indexes of bronchiolar injury, whereas all groups exhibited an increased number of polymorphonuclear leukocytes in alveolar septa, which was significantly greater in group C. In conclusion, administration of exogenous surfactant largely prevents the histological and functional damage of prolonged MV at low lung volumes, whereas surfactant dysfunction worsens the functional alterations, also because of edema formation and, possibly, increased inflammatory response.