Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels

Most known organisms encode proteases that are crucial for constitutive proteolytic events. In the present paper, we describe a method to define these events in proteomes from Escherichia coli to humans. The method takes advantage of specific N-terminal biotinylation of protein samples, followed by affinity enrichment and conventional LC (liquid chromatography)-MS/MS (tandem mass spectrometry) analysis. The method is simple, uses conventional and easily obtainable reagents, and is applicable to most proteomics facilities. As proof of principle, we demonstrate profiles of proteolytic events that reveal exquisite in vivo specificity of methionine aminopeptidase in E. coli and unexpected processing of mitochondrial transit peptides in yeast, mouse and human samples. Taken together, our results demonstrate how to rapidly distinguish real proteolysis that occurs in vivo from the predictions based on in vitro experiments.     

Premature aging in uremia

Guanidinosuccinic acid is an aberrant metabolite isolated 40 years ago in the blood and urine of uremic subjects and a suspect in the toxicity associated with renal failure. It plays a minor role in the bleeding diathesis of uremia, contributes to the methyl group deficiency of dialysis patients, and is a factor in the premature atherosclerosis of end stage renal disease through the induction of hyperhomocysteinemia. As a major player, however, in the diversity and severity of uremic symptoms, it is a disappointment. Recently its source has been identified. It results from the superoxidation of argininosuccinic acid, which leads, also, to the production of gamma glutamic semialdehyde, an advanced glycation end product (AGE), which normally results from from the Maillard reaction, the non-enzymatic browning of protein. AGEs stimulate cross-linkages in protein that lead ultimately to loss of function, phagocytosis, and removal, and are important elements in the premature aging characteristic of renal disease, and diabetes.     

Real time computation: zooming in on population codes

Information processing in nervous systems intricately combines computation at the neuronal and network levels. Many computations may be envisioned as sequences of signal processing steps along some pathway. How can information encoded by single cells be mapped onto network population codes, and how do different modules or layers in the computation synchronize their communication and computation? These fundamental questions are particularly severe when dealing with real time streams of inputs. Here we study this problem within the context of a minimal signal perception task. In particular, we encode neuronal information by externally applying a space- and time-localized stimulus to individual neurons within a network. We show that a pulse-coupled recurrent neural network can successfully handle this task in real time, and obeys three key requirements: (i) stimulus dependence, (ii) initial-conditions independence, and (iii) accessibility by a readout mechanism. In particular, we suggest that the network's overall level of activity can be used as a temporal cue for a robust readout mechanism. Within this framework, the network can rapidly map a local stimulus onto a population code that can then be reliably read out during some narrow but well defined window of time.     

Oxidized cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation

Apolipoprotein amyloid deposits and lipid oxidation products are colocalized in human atherosclerotic tissue. In this study we show that the primary ozonolysis product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (KA), rapidly promotes human apolipoprotein (apo) C-II amyloid fibril formation in vitro. Previous studies show that hydrophobic aldehydes, including KA, modify proteins by the formation of a Schiff base with the lysine epsilon-amino group or N-terminal amino group. High-performance liquid chromatography, mass spectrometry, and proteolysis of KA-modified apoC-II revealed that KA randomly modified six different lysine residues, with primarily one KA attached per apoC-II molecule. Competition experiments showed that an aldehyde scavenging compound partially inhibited the ability of KA to hasten apoC-II fibril formation. Conversely, the acid derivative of KA, lacking the ability to form a Schiff base, accelerated apoC-II fibril formation, albeit to a lesser extent, suggesting that amyloidogenesis triggered by KA involves both covalent and noncovalent mechanisms. The viability of a noncovalent mechanism mediated by KA has been observed previously with alpha-synuclein aggregation, implicated in Parkinson's disease. Electron microscopy demonstrated that fibrils formed in the presence of KA had a similar morphology to native fibrils; however, the isolated KA-apoC-II covalent adducts in the absence of unmodified apoC-II formed fibrillar structures with altered ropelike morphologies. KA-mediated fibril formation by apoC-II was inhibited by the addition of the amine-containing compound hydralazine and the lipid-binding protein apoA-I. These in vitro studies suggest that the oxidized small molecule pool could trigger or hasten the aggregation of apoC-II to form amyloid deposits.     

Identification and synthesis of major metabolites of Vasopressin V2-receptor agonist WAY-151932, and antagonist, Lixivaptan

Small molecule agonists and antagonists of the V(2)-vasopressin receptor have been discovered and have undergone clinical trials. In conjunction with these discovery programs, the synthesis and biological testing of various metabolites associated with these clinical targets were actively pursued. We now report the results of our synthetic efforts and the corresponding biological data generated for several of the metabolites of WAY-151932 and CL-347985 (Lixivaptan).     

Biological solutions to transport network design

Transport networks are vital components of multicellular organisms, distributing nutrients and removing waste products. Animal and plant transport systems are branching trees whose architecture is linked to universal scaling laws in these organisms. In contrast, many fungi form reticulated mycelia via the branching and fusion of thread-like hyphae that continuously adapt to the environment. Fungal networks have evolved to explore and exploit a patchy environment, rather than ramify through a three-dimensional organism. However, there has been no explicit analysis of the network structures formed, their dynamic behaviour nor how either impact on their ecological function. Using the woodland saprotroph Phanerochaete velutina, we show that fungal networks can display both high transport capacity and robustness to damage. These properties are enhanced as the network grows, while the relative cost of building the network decreases. Thus, mycelia achieve the seemingly competing goals of efficient transport and robustness, with decreasing relative investment, by selective reinforcement and recycling of transport pathways. Fungal networks demonstrate that indeterminate, decentralized systems can yield highly adaptive networks. Understanding how these relatively simple organisms have found effective transport networks through a process of natural selection may inform the design of man-made networks.     

Association between plasmid carrying an expanded-spectrum cephalosporin resistance and biofilm formation in Escherichia coli

The formation of biofilm is a universal bacterial survival strategy. Biofilms occur on inert and living support in the natural environment and in industrial installations. This microenvironment leads to the horizontal transfer of genetic material between bacteria by physical contact. In order to evaluate the relationship between biofilm-forming capabilities, surface characteristics and plasmid content we purified from Salmonella a plasmid conferring resistance to cephalosporin and transferred it by electroporation to E.coli DH10B originally unable to form biofilm in inert surface. We demonstrated the association between a plasmid conferring resistance to expanded-spectrum cephalosporin and biofilm formation. We also noted that this plasmid influences the cell surface properties and cell motility.