Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of carbohydrate moieties of cholecystokinin receptors in cholecystokinin octapeptide binding: alteration of binding data by specific lectins

Cholecystokinin (CCK) receptors on rat pancreatic acini have been demonstrated to be glycoproteins. In order to study whether their carbohydrate moieties play a role in ligand binding, membrane preparations (adjusted to 0.2 mg protein me) were incubated with 20 pM 125-I-CCK octapeptide (125I-CCK8) for 4 h at 30 degrees C in the presence of lectins with different sugar specificities. Concanavalin A, soy-bean agglutinin, and peanut agglutinin in concentrations up to 1 mM did not alter specific 125I-CCK8 binding. Ulex europeus lectin I showed a dose-dependent enhancement of CCK binding up to 150% of controls at a concentration of 1 mM. Wheat-germ agglutinin (WGA) was the only lectin found to have an inhibitory effect. Inhibition was dose-dependent, with maximal reduction attained at 42 nM, but CCK binding was only partially inhibited to 66.2 +/- 4.4%. Inhibition by WGA was prevented by the presence of N-acetyl-D-glucosamine or N,N',N"-triacetylchitotriose, sugars that are specific for WGA. The inhibitory effect of WGA was not due to an increase in non-specific binding, increased CCK degradation, or CCK binding to WGA. Binding data indicated that the presence of WGA resulted in a decrease in receptor affinity (Kd = 567 +/- 191 v. 299 +/- 50 pM). No significant change in the number of available binding sites was observed. This suggests that WGA is not binding to the active binding site. It is conceivable that binding of WGA to N-acetyl-D-glucosamine or its polymers can lead to a conformational change in the receptor protein, and that this carbohydrate moiety is essential for optimal receptor-ligand interaction.     

Discrete modeling for integration and analysis of large-scale signaling networks

Most biological processes are orchestrated by large-scale molecular networks which are described in large-scale model repositories and whose dynamics are extremely complex. An observed phenotype is a state of this system that results from control mechanisms whose identification is key to its understanding. The Biological Pathway Exchange (BioPAX) format is widely used to standardize the biological information relative to regulatory processes. However, few modeling approaches developed so far enable for computing the events that control a phenotype in large-scale networks.Here we developed an integrated approach to build large-scale dynamic networks from BioPAX knowledge databases in order to analyse trajectories and to identify sets of biological entities that control a phenotype. The Cadbiom approach relies on the guarded transitions formalism, a discrete modeling approach which models a system dynamics by taking into account competition and cooperation events in chains of reactions. The method can be applied to every BioPAX (large-scale) model thanks to a specific package which automatically generates Cadbiom models from BioPAX files.The Cadbiom framework was applied to the BioPAX version of two resources (PID, KEGG) of the Pathway Commons database and to the Atlas of Cancer Signalling Network (ACSN). As a case-study, it was used to characterize sets of biological entities implicated in the epithelial-mesenchymal transition. Our results highlight the similarities between the PID and ACSN resources in terms of biological content, and underline the heterogeneity of usage of the BioPAX semantics limiting the fusion of models that require curation. Causality analyses demonstrate the smart complementarity of the databases in terms of combinatorics of controllers that explain a phenotype. From a biological perspective, our results show the specificity of controllers for epithelial and mesenchymal phenotypes that are consistent with the literature and identify a novel signature for intermediate states.     

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

Following the recovery of naso-pharyngeal cancer patients by trace elements in hair using statistical pattern recognition methods

This article describes a study where the recovery of nasopharyngeal cancer (NPC) patients was traced by trace elements (TEs) in hair of these patients. These patients whose concentrations of TEs in hair were studied are volunteers who were divided into three groups B, C, and D. Group B was made up of volunteers who had just been diagnosed as having NPC, group C and group D were made up of volunteers who had been diagnosed as having NPC after 3 and 6 mo, respectively, of treatment. For comparison, a control group, namely group A, which was made up of volunteers from healthy persons was added to this study. By implementation of statistic pattern recognition methods, it has been found that the concentrations of TEs in hair can remarkably reflect different recovery phases of NPC patients.     

Green fluorescent protein-tagged Edwardsiella tarda reveals portal of entry in fish

The application of green fluorescent protein (GFP) to identify the portal(s) of entry of bacterial pathogens in animal hosts was studied using the fish pathogen Edwardsiella tarda and blue gourami, Trichogaster trichopterus. An immersion challenge model was utilized to mimic natural infection conditions in fish. Gastrointestinal tract, gills and the body surface of fish were found to be the sites of entry of virulent E. tarda (PPD130/91) by histological and infection kinetics studies. On the other hand, avirulent E. tarda (PPD125/87) was mainly found in the gastrointestinal tract, and the bacterial population in tissue declined over a period of 7 days.