Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain

Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholerae that increases intestinal permeability by interacting with a mammalian cell receptor with subsequent activation of intracellular signaling leading to the disassembly of the intercellular tight junctions. Zot localizes in the bacterial outer membrane of V. cholerae with subsequent cleavage and secretion of a carboxyl-terminal fragment in the host intestinal milieu. To identify the Zot domain(s) directly involved in the protein permeating effect, several zot gene deletion mutants were constructed and tested for their biological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures. The Zot biologically active domain was localized toward the carboxyl terminus of the protein and coincided with the predicted cleavage product generated by V. cholerae. This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation. Amino acid comparison between the Zot active fragment and zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot.     

Spin label electron paramagnetic resonance study in thylakoid membranes from a new herbicide-resistant D1 mutant from soybean cell cultures deficient in fatty acid desaturation

We examined the effect of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the production of hydroxyl radical (*OH) generation via nitric oxide synthase (NOS) activation by an in vivo microdialysis technique. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and tissue was perfused with Ringer's solution through the microdialysis probe at a rate of 1 microl/min. Sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused directly through a microdialysis probe to detect the generation of *OH. Induction of [K(+)](o) (70 mM) or tyramine (1 mM), significantly increased the formation of *OH trapped as 2,3-dihydroxybenzoic acid (DHBA). The application of N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, significantly decreased the K(+) depolarization-induced *OH formation, but the effect of tyramine significantly increased the level of 2,3-DHBA. When fluvastatin (100 microM), an inhibitor of low-density lipoprotein (LDL) oxidation, was administered to L-NAME-pretreated animals, both KCl and tyramine failed to increase the level of 2,3-DHBA formation. The effect of fluvastatin may be unrelated to K(+) depolarization-induced *OH generation. To examine the effect of fluvastatin on ischemic/reperfused rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery (LAD). When the heart was reperfused, a marked elevation of the level of 2,3-DHBA was observed. However, in the presence of fluvastatin (100 microM), the elevation of 2,3-DHBA was not observed in ischemia/reperfused rat heart. Fluvastatin, orally at a dose of 3 mg/kg/day for 4 weeks, significantly blunted the rise of serum creatine phosphokinase and improved the electrocardiogram 2 h after coronary occlusion. These results suggest that fluvastatin is associated with a cardioprotective effect due to the suppression of noradrenaline-induced *OH generation by inhibiting LDL oxidation in the heart.     

Poster Sessions AP01: Gene Expression and Regulation. Regulation of gene expression during the development of cortico‐thalamic connection in rat

The formation of Cortico‐Thalamic projections requires the precise spatial and temporal expression of proteins that are involved in the different stages of synaptogenesis. We reasoned that the underlying molecular mechanism of this process is the differential expression of genes that code for stage specific proteins. Our research objective was to identify the differential expressed mRNAs during the main stages of synapses formation, which starts at embryonic day 12 (E12) and finishes on the first postnatal days in the rat. We approach this problem using Differential Display technique on three distinct ages of rat cerebral cortex that were: E13, E18 and postnatal day 0 (P0). We found 80 differential bands using 54 random primers and 18 of them were cloned and sequenced. The sequence analysis showed among others, a cDNA fragment highly homologous with the human A Kinase Anchoring Protein 450/350 also called CG‐NAP. We found that this cDNA fragment homologous to AKAP was up regulated at E15 when cortical cells are undergoing active axogenesis. The expression pattern of this cDNA was confirmed by Real Time PCR. Our findings suggest a possible function for AKAP 450 in the regulation of the state of phosphorylation of centrosomal components during the initial stages of synapses formation during the establishment of Cortico‐Thalamic connection.     

Anti-inflammatory constituents of Mortonia greggii Gray

A new phytochemical study of Mortonia greggii (Celastraceae) afforded four friedelan derivatives (1-4), three lupanes (5-7), retusine (8), two esterified polyhydroxyagarofurans (9-10), mortonin C (11) and photomortonin C (12). The anti-inflammatory activity on carrageenan and 12-O-tetradecanoylphorbol-13-acetate induced models of inflammation, as well as the ability to inhibit the nitric oxide (NO) produced by lipopolysaccharide-stimulated mouse peritoneal macrophages were evaluated for the main metabolites. Our results showed that the friedelan dehydrocanophyllic acid methyl ester (1) exhibits an anti-inflammatory effect which could be related to an inhibition of prostaglandin and NO production. The activity of lupeol (5), 29-hydroxylupeol (6) and 29-hydroxylupenone (7) might be involved with the prostanoid synthesis. The presence of the hydroxy groups in 6 appears to be important for activity. The edema inhibition capacity of retusine (8) could be related to a reduction of the prostaglandin production. The agarofuran derivative 10 is an NO inhibitor whose activity is probably not involved in the synthesis of prostaglandins.     

Nitrosylation of rabbit ferrous heme-hemopexin

Hemopexin (HPX) serves as a trap for toxic plasma heme, ensuring its complete clearance by transportation to the liver. Moreover, HPX-heme has been postulated to play a key role in the homeostasis of nitric oxide (NO). Here, the thermodynamics for NO binding to rabbit ferrous HPX-heme as well as the EPR and optical absorption spectroscopic properties of rabbit ferrous nitrosylated HPX-heme (HPX-heme-NO) are reported. The value of the dissociation equilibrium constant for NO binding to rabbit ferrous HPX-heme (i.e., H) is (1.4±0.2)×10^–7 M, at pH 7.0 and 10.0 °C; the value of H is unaffected by sodium chloride. At pH 7.0, rabbit ferrous HPX-heme-NO is a six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry and by =146 mM^–1 cm^–1 at 419 nm. At pH 4.0, rabbit ferrous HPX-heme-NO is a five-coordinate heme-iron species, characterized by an X-band EPR spectrum with three-line splitting centered at 334 mT and by =74 mM^–1 cm^–1 at 387 nm. The pK_a value of the reversible pH-induced six- to five-coordinate spectroscopic transition is 4.8±0.1 in the absence of sodium chloride and 4.3±0.1 in the presence of 1.5×10^–1 M sodium chloride. This result is in agreement with the effect of sodium chloride on rabbit HPX-heme stability. The present data have been analyzed in parallel with those of a related heme model compound and heme-protein systems.     

YAdumper: extracting and translating large information volumes from relational databases to structured flat files

Downloading the information stored in relational databases into XML and other flat formats is a common task in bioinformatics. This periodical dumping of information requires considerable CPU time, disk and memory resources. YAdumper has been developed as a purpose-specific tool to deal with the integral structured information download of relational databases. YAdumper is a Java application that organizes database extraction following an XML template based on an external Document Type Declaration. Compared with other non-native alternatives, YAdumper substantially reduces memory requirements and considerably improves writing performance.     

SQUARE--determining reliable regions in sequence alignments

The Server for Quick Alignment Reliability Evaluation (SQUARE) is a Web-based version of the method we developed to predict regions of reliably aligned residues in sequence alignments. Given an alignment between a query sequence and a sequence of known structure, SQUARE is able to predict which residues are reliably aligned. The server accesses a database of profiles of sequences of known three-dimensional structures in order to calculate the scores for each residue in the alignment. SQUARE produces a graphical output of the residue profile-derived alignment scores along with an indication of the reliability of the alignment. In addition, the scores can be compared against template secondary structure, conserved residues and important sites.     

SPOC: A widely distributed domain associated with cancer,apoptosis and transcription

Background  

The Split ends (Spen) family are large proteins characterised by N-terminal RNA recognition motifs (RRMs) and a conserved SPOC (Spen paralog and ortholog C-terminal) domain. The aim of this study is to characterize the family at the sequence level.