Mechanism of thrombin clearance by human astrocytoma cells

Astroglial cells secrete a variety of factors that contribute to the regulation of neurite initiation and continued outgrowth, among them proteases and protease inhibitors. An alteration in the balance between these proteins has been implicated in Alzheimer's disease, resulting in an accumulation of thrombin:protease nexin 1 (PN1) complexes in the brains of these patients. This report aims at providing a biochemical explanation for this phenomenon. We show that human astrocytoma cells bind and internalize thrombin and thrombin:PN1 complexes efficiently by a PN1-dependent mechanism. Binding was potently inhibited by soluble heparin and did not occur with the mutant PN1 (K7E) deficient in heparin binding. Receptor-associated protein, an antagonist of the low-density lipoprotein receptor-related protein (LRP), inhibited internalization of thrombin by the astrocytoma cells, but did not affect cell-surface binding. The results are consistent with a mechanism by which astrocytoma cells clear thrombin in a sequential manner: thrombin is first complexed with PN1, then bound to cell-surface heparins, and finally internalized by LRP. This mechanism provides a link between the neuronal growth regulators thrombin and PN1 and proteins genetically associated with Alzheimer's disease, such as LRP.     

Mechanism of oxidation of pi bonds by cytochrome P-450. Electronic requirements of the transition state in the turnover of phenylacetylenes

Substituted phenylacetylenes are oxidized by purified reconstituted rat liver cytochrome P-450b to the corresponding 2-arylacetic acids. A linear free energy correlation with a rho value of -2.2 exists between the rates of formation of the arylacetic acids and the substituent sigma constants. The enzyme is simultaneously inactivated but the inactivation rates, in contrast to those of metabolite formation, are essentially identical for all the phenylacetylenes. Inactivation is thus insensitive to the electronic nature of the substituents. The metabolite: inactivation ratio consequently decreases from 38 for p-methylphenylacetylene to 4 for p-nitrophenylacetylene. Replacement of the terminal proton of phenylacetylene with deuterium gives rise to an isotope effect of 1.7 on metabolite formation without altering enzyme inactivation. The differential effect of substituents on heme alkylation and metabolite formation requires the two pathways to diverge early in the catalytic process and argues against mechanisms that involve radical cation intermediates.     

Development and application of the active surveillance of pathogens microarray to monitor bacterial gene flux

Background  

Human and animal health is constantly under threat by emerging pathogens that have recently acquired genetic determinants that enhance their survival, transmissibility and virulence. We describe the construction and development of an Active Surveillance of Pathogens (ASP) oligonucleotide microarray, designed to 'actively survey' the genome of a given bacterial pathogen for virulence-associated genes.     

The structure, dynamics, and binding of the LA45 module pair of the low-density lipoprotein receptor suggest an important role for LA4 in ligand release

The low-density lipoprotein receptor (LDLR), the primary receptor for cholesterol uptake, binds ligands through its seven LDL-A modules (LAs). We present nuclear magnetic resonance (NMR) and ligand binding measurements on the fourth and fifth modules of the LDLR (LA45), the modules critical for ApoE binding, at physiological pH. Unlike LA5 and all other modules in LDLR, LA4 has a very weak calcium affinity, which probably plays a critical role in endosomal ligand release. The NMR solution structure of each module in the LA45 pair only showed minor differences compared to the analogous domains in previously determined crystal structures. The 12-residue linker connecting the modules, though slightly structured through an interaction with LA4, is highly flexible. Although no intermodule nuclear Overhauser effects were detected, chemical shift perturbations and backbone dynamics suggest cross talk between the two modules. The ligand affinity of both modules is enhanced when the two are linked. LA4 is more flexible than LA5 and remains so even in the module pair, which likely is related to its weaker calcium binding affinity.     

Trypsinogen activation as observed in accelerated molecular dynamics simulations

Serine proteases are involved in many fundamental physiological processes, and control of their activity mainly results from the fact that they are synthetized in an inactive form that becomes active upon cleavage. Three decades ago Martin Karplus's group performed the first molecular dynamics simulations of trypsin, the most studied member of the serine protease family, to address the transition from the zymogen to its active form. Based on the computational power available at the time, only high frequency fluctuations, but not the transition steps, could be observed. By performing accelerated molecular dynamics (aMD) simulations, an interesting approach that increases the configurational sampling of atomistic simulations, we were able to observe the N‐terminal tail insertion, a crucial step of the transition mechanism. Our results also support the hypothesis that the hydrophobic effect is the main force guiding the insertion step, although substantial enthalpic contributions are important in the activation mechanism. As the N‐terminal tail insertion is a conserved step in the activation of serine proteases, these results afford new perspective on the underlying thermodynamics of the transition from the zymogen to the active enzyme.     

Evolutionary divergence times in the Annonaceae: evidence of a late Miocene origin of Pseuduvaria in Sundaland with subsequent diversification in New Guinea

Background  

Phylogenetic analyses of the Annonaceae consistently identify four clades: a basal clade consisting of Anaxagorea, and a small 'ambavioid' clade that is sister to two main clades, the 'long branch clade' (LBC) and 'short branch clade' (SBC). Divergence times in the family have previously been estimated using non-parametric rate smoothing (NPRS) and penalized likelihood (PL). Here we use an uncorrelated lognormal (UCLD) relaxed molecular clock in BEAST to estimate diversification times of the main clades within the family with a focus on the Asian genus Pseuduvaria within the SBC. Two fossil calibration points are applied, including the first use of the recently discovered Annonaceae fossil Futabanthus. The taxonomy and morphology of Pseuduvaria have been well documented, although no previous dating or biogeographical studies have been undertaken. Ancestral areas at internal nodes within Pseuduvaria are determined using dispersal-vicariance analysis (DIVA) and weighted ancestral area analysis (WAAA).     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.     

Thermal plasticity in Drosophila melanogaster : A comparison of geographic populations

Background  

Populations of Drosophila melanogaster show differences in many morphometrical traits according to their geographic origin. Despite the widespread occurrence of these differences in more than one Drosophila species, the actual selective mechanisms controlling the genetic basis of such variation are not fully understood. Thermal selection is considered to be the most likely cause explaining these differences.