A genetic analysis of aromatic amino acid hydroxylases involvement in DOPA synthesis during Drosophila adult development

This study was designed to examine whether cyclosporine (CyA) acts on the endocrine system by modifying the pulsatile secretion pattern of prolactin and LH. Both pituitary-grafted and sham-operated rats were submitted to a subcutaneous vehicle or CyA (5 mg/kg) treatment daily for 10 days beginning on the day of surgery. Pituitary grafting and/or CyA administration changed the pulsatile pattern of prolactin observed in sham-operated animals. The mean values of serum prolactin were significantly increased by pituitary grafting and the treatment with CyA further increased them. The mean half-life of prolactin was significantly increased in pituitary-grafted rats and was not changed by CyA administration, although it was decreased in sham-operated rats. The frequency of prolactin pulses was significantly decreased in pituitary-grafted as compared to sham-operated controls and was not further modified by CyA administration. However, in sham-operated rats a significant decrease of this parameter was observed. Duration of the prolactin peaks was significantly increased by pituitary grafting, and was not modified by CyA administration in any of the groups studied. The absolute amplitude of the prolactin peaks was significantly increased in pituitary-grafted as compared to sham-operated animals, and the treatment with CyA further increased this parameter in both groups. Mean values of LH were significantly increased in pituitary-grafted as compared to control rats. CyA administration significantly increased LH levels in sham-operated rats and decreased them in pituitary-grafted animals. The mean half-life, the pulse frequency and the duration of LH peaks were not modified by either pituitary grafting or CyA administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Li influx and binding, and li/mg competition in bovine chromaffin cell suspensions as studied by li NMR and fluorescence spectroscopy

Li(+) influx by bovine chromaffin cells, obtained from bovine adrenal medulla, was studied in intact cell suspensions using (7)Li NMR spectroscopy with the shift reagent [Tm(HDOTP)](4-). The influx rate constants, k(i), were determined in the absence and in the presence of two Na(+) membrane transport inhibitors. The values obtained indicate that both voltage sensitive Na(+) channels and (Na(+)/K(+))-ATPase play an important role in Li(+) uptake by these cells. (7)Li NMR T(1) and T(2) relaxation times for intracellular Li(+) in bovine chromaffin cells provided a T(1)/T(2) ratio of 305, showing that Li(+) is highly, immobilized due to strong binding to intracellular structures. Using fluorescence spectroscopy and the Mg(2+) fluorescent probe, furaptra, the free intracellular Mg(2+) concentration in the bovine chromaffin cells incubated with 15 mM LiCl was found to increase by about mM after the intracellular Li(+) concentration reached a steady state. Therefore, once inside the cell, Li(+) is able to displace Mg(2+) from its binding sites.     

Twister: the development of a peer-to-peer microblogging platform

This paper proposes a new microblogging architecture based on peer-to-peer networks overlays. The proposed platform comprises three mostly independent overlay networks. The first provides distributed user registration and authentication and is based on the Bitcoin protocol. The second one is a Distributed Hash Table (DHT) overlay network providing key/value storage for user resources and tracker location for the third network. The last network is a collection of possibly disjoint ‘swarms’ of followers, based on the BitTorrent protocol, which can be used for efficient near-instant notification delivery to many users. By leveraging existing and proven technologies, twister provides a new microblogging platform offering security, scalability and privacy features. A built-in mechanism provides incentive for entities that contribute processing time to run the user registration network, rewarding such entities with the privilege of sending a single unsolicited (‘promoted’) message to the entire network. The number of unsolicited messages per day is defined in order to not upset users. Security and scalability assumptions have been confirmed in a real-world deployment.     

Towards a smooth design process for static communicative node‐link diagrams

Node‐link infographics are visually very rich and can communicate messages effectively, but can be very difficult to create, often involving a painstaking and artisanal process. In this paper we present an investigation of node‐link visualizations for communication and how to better support their creation. We begin by breaking down these images into their basic elements and analyzing how they are created. We then present a set of techniques aimed at improving the creation workflow by bringing more flexibility and power to users, letting them manipulate all aspects of a node‐link diagram (layout, visual attributes, etc.) while taking into account the context in which it will appear. These techniques were implemented in a proof‐of‐concept prototype called GraphCoiffure, which was designed as an intermediary step between graph drawing/editing software and image authoring applications. We describe how GraphCoiffure improves the workflow and illustrate its benefits through practical examples.     

Hierarchical classification of protein function with ensembles of rules and particle swarm optimisation

This paper focuses on hierarchical classification problems where the classes to be predicted are organized in the form of a tree. The standard top-down divide and conquer approach for hierarchical classification consists of building a hierarchy of classifiers where a classifier is built for each internal (non-leaf) node in the class tree. Each classifier discriminates only between its child classes. After the tree of classifiers is built, the system uses them to classify test examples one class level at a time, so that when the example is assigned a class at a given level, only the child classes need to be considered at the next level. This approach has the drawback that, if a test example is misclassified at a certain class level, it will be misclassified at deeper levels too. In this paper we propose hierarchical classification methods to mitigate this drawback. More precisely, we propose a method called hierarchical ensemble of hierarchical rule sets (HEHRS), where different ensembles are built at different levels in the class tree and each ensemble consists of different rule sets built from training examples at different levels of the class tree. We also use a particle swarm optimisation (PSO) algorithm to optimise the rule weights used by HEHRS to combine the predictions of different rules into a class to be assigned to a given test example. In addition, we propose a variant of a method to mitigate the aforementioned drawback of top-down classification. These three types of methods are compared against the standard top-down hierarchical classification method in six challenging bioinformatics datasets, involving the prediction of protein function. Overall HEHRS with the rule weights optimised by the PSO algorithm obtains the best predictive accuracy out of the four types of hierarchical classification method.     

Determinants of the inhibitory action of purified 14-kDa phospholipases A2 on cell-free prothrombinase complex

It has been suggested (Kini, R. R., and Evans, H. J. (1987) J. Biol. Chem. 262, 14402-14407) that the anticoagulant activity of members of the 14-kDa phospholipase A2 (PLA2) family depends on the presence of basic residues within a variable surface region (residues 54-77) distinct from both the conserved catalytic machinery and surface sites mediating the antibacterial action of these enzymes (see Weiss, J., Inada, M., Elsbach, P., and Crowl, R. M. (1994) J. Biol. Chem. 269, 26331-26337). To further define the determinants of the anticoagulant activity of PLA2, we have analyzed the inhibitory effects of purified native and recombinant PLA2 on cell-free prothrombinase. Both native and recombinant wild-type pig pancreas (net charge -1) and human "secretory" PLA2 (net charge +15) produced similar dose-dependent inhibition of prothrombinase activity that was significantly less potent than a toxic PLA2 purified from snake venom. Site-specific mutations that either increased or decreased PLA2 activity toward bactericidal/permeability-increasing protein-treated Escherichia coli by up to 50-fold had no effect on antiprothrombinase activity. In contrast, substitution of Arg for Asp-59/Gly for Ser-60 in the pig PLA2 increased antiprothrombinase activity by 5-10-fold without affecting catalytic activity toward a range of phospholipid substrates or antibacterial activity. Comparison of antiprothrombinase activity of catalytically active and inactive forms of the PLA2 and under a range of phospholipid conditions revealed that the potent antiprothrombinase activity of native toxic venom PLA2 and of the D59R.S60G mutant pancreatic PLA2 reflect combined catalytic and noncatalytic actions, the latter apparently dependent on basic residues at discrete surface sites in the enzyme.     

Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin

We generated a series of libraries having variants of the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1, also known as tissue-factor pathway inhibitor-I) displayed on bacteriophage M13 as pIII-fusions. We varied LACI-DI iteratively in two regions: the P1 region (positions 10-21) and the "second loop", (positions 31-39), which together form one end of the domain. Display-phage library Lib#1 allows 31 200 amino-acid sequences in P1 region (residues 13, 16-19). Preliminary, we screened Lib#1 against human plasmin (PLA, EC 3.4.21.7) immobolized on agarose to enrich for phage displaying variants with PLA affinity. We introduced a 1600-fold increase in second-loop diversity (residues 31, 32, 34, 39) into the population of selectants from Lib#1, yielding Lib#2. Lib#2 (allowing approximately 50 million amino-acid sequences) was screened against PLA-agarose to isolate highest affinity binders. Protein EPI-P211, derived from the best isolate of Lib#2, inhibits PLA with Ki = 2 nM (at least 500-fold better than LACI-D1) and with high specificity. We used amino-acid sequences of PLA-binding selectants to design a PLA-biased library (Lib#3) which we screened against PLA. The protein EPI-P302 (derived from the best binder obtained from Lib#3) has Ki for PLA inhibition of 87 pM, which is 25-fold better than the first-round best binder and > or = 12 500-fold better than LACI-D1. EPI-P302 also shows very high specificity for PLA vs other human proteases and is resistant to inactivation by oxidants and extremes of temperature or pH. Thus, one can use selectants from one library to design target-tailored combinatorial libraries and obtain quite stable, highly specific, very high-affinity binding molecules while maintaining an essentially human framework.