Prediction of protein secondary structure by neural networks: encoding short and long range patterns of amino acid packing.

A complex, cascaded neural network designed to predict the secondary structure of globular proteins has been developed. Information about the local buried-unburied pattern and the average tendency of the particular types of amino acids to be buried inside the globule were used. Nonspecific information about long distance contact maps was also employed. These modifications result in a noticeable improvement (3-9%) of prediction accuracy. The best result for the average success ratio for the testing set of nonhomologous proteins was 68.3% (with corresponding Matthews' coefficients, C alpha,beta,coil equal to 0.60, 0.47, 0.43, respectively).     

Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells

Cationic liposomes can efficiently carry nucleic acids into mammalian cells. This property is tightly connected with their ability to fuse with negatively charged natural membranes (i.e. the plasma membrane and endosomal membrane). We used FRET to monitor and compare the efficiency of lipid mixing of two liposomal preparations — one of short-chained diC14-amidine and one of long-chained unsaturated DOTAP — with the plasma membrane of HeLa cells. The diC14-amidine liposomes displayed a much higher susceptibility to lipid mixing with the target membranes. They disrupted the membrane integrity of the HeLa cells, as detected using the propidium iodide permeabilization test. Morphological changes were transient and essentially did not affect the viability of the HeLa cells. The diC14-amidine liposomes were much more effective at either inducing lipid mixing or facilitating transfection.     

Human DHHC proteins: a spotlight on the hidden player of palmitoylation

Palmitoylation is one of the most common posttranslational lipid modifications of proteins and we now know quite a lot about it. However, the state of knowledge about the enzymes that catalyze this process is clearly insufficient. This review is focused on 23 human DHHC genes and their products - protein palmitoyltransferases. Here we describe mainly the structure and function of these proteins, but also, to a lesser degree, what the substrates of the enzymes are and whether they are related to various diseases. The main aim of this review was to catalogue existing information concerning the human DHHC family of genes/proteins, making them and their functions easier to understand.     

Pescadillo interacts with the cadmium response element of the human heme oxygenase-1 promoter in renal epithelial cells

Renal tubular cells elicit adaptive responses following exposure to nephrotoxins, such as cadmium. One response is the up-regulation of the 32-kDa redox-sensitive protein, heme oxygenase-1. Exposure of renal proximal tubular epithelial cells to 10 mum cadmium demonstrated induction ( approximately 20-fold) of heme oxygenase-1 mRNA and protein. Using a 4.5-kb human heme oxygenase-1 promoter construct, the importance of a previously identified cadmium response element (TGCTAGAT) in HeLa cells was verified in renal epithelial cells. Specific protein-DNA interaction with this sequence was demonstrated using nuclear extracts from cadmium-treated cells. Yeast one-hybrid screen of a human kidney cDNA library resulted in the identification of pescadillo, a unique nucleolar, developmental protein, as an interacting protein with the cadmium response element and was confirmed by chromatin immunoprecipitation in vivo and gel shift assays with purified glutathione S-transferase-pescadillo protein in vitro. The specificity of the DNA-protein interaction was verified by the absence of a binding complex when the core sequence of the cadmium response element was mutated or deleted. In addition, B23/nucleophosmin, another nucleolar protein, did not interact with the cadmium response sequence. Overexpression of pescadillo resulted in increased activity of the 4.5-kb human heme oxygenase-1 promoter construct but failed to activate this construct when the cadmium response sequence was mutated. The findings demonstrate the important and previously unrecognized role of pescadillo as a DNA-binding protein interacting specifically with the cadmium response element of the human heme oxygenase-1 gene.     

Preparation, single-crystal X-ray diffraction and high-resolution NMR spectroscopic analyses of N-[(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D,L-ribitol)-5-yl]trimethylammonium iodide

The synthesis and isolation of 1,4-anhydro-5-deoxy-5-iodo-2,3-O-isopropylidene-D,L-ribitol and N-[(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D,L-ribitol)-5-yl]trimethylammonium iodide are described. The products were examined by (1)H, (13)C NMR spectroscopy, and N-[(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D,L-ribitol)-5-yl]trimethylammonium iodide was additionally analyzed by X-ray crystallography.     

PS exposure increases the susceptibility of cells to fusion with DOTAP liposomes

Cationic liposomes are used as efficient carriers for gene delivery into mammalian cells due to their ability to bind nucleic acids, adsorb onto the cell surface and fuse with negatively charged membranes. This last property enables the release and escape of their cargo from endosomal compartments. The efficiency of this fusion mainly depends on the surface charge of the target membranes. Here, we report that cells of two different lines, epithelial adenocarcinoma HeLa and lymphocytic leukemia Jurkat T, which externalize PS, are more susceptible to fusion with DOTAP liposomes than control cells. We compared the ability to undergo fusion of untreated and apoptotic cells. Apoptosis was induced by various pro-apoptotic agents and treatments, namely: incubation in the presence of MnCl(2), cytostatic drugs fludarabine and mitoxantrone, staurosporine and serum depletion in the case of HeLa cells. Jurkat T cells were treated similarly except apoptosis was additionally induced by incubation in the presence of 4% EtOH. Epithelial cells fused with the highest efficiencies of lipid mixing, when pretreated with staurosporine. Jurkat T cells were less susceptible to fusion, but they also displayed an increase in fusion efficiency after the induction of apoptosis. Alternatively, we treated the cells with metabolic inhibitors causing ATP-depletion in order to inactivate aminophospholipid translocase. After ATP-depletion, HeLa and Jurkat T cells fused with DOTAP liposomes with higher efficiencies than control cells. Our conclusion is that the lipid asymmetry of natural membranes may limit fusion with cationic liposomes.     

Selection in a cycling population: differential response among skeletal traits

Abstract Population density cycles influence phenotypic evolution through both density‐dependent selection during periods of high density and through enhanced genetic drift during periods of low density. We investigated the response of different phenotypic traits to the same density cycles in a population of the yellow‐necked mouse, Apodemus flavicollis, from Bia?owieza National Park in Poland. We examined nonmetric skull traits, skull and mandible size, skull and mandible shape, and transferrin allele frequencies. We found that all of the traits changed significantly over the seven‐year study period. The greatest changes in nonmetric traits and mandible size occurred during periods of increasing density, and the magnitude of changes in skull and mandible shape was correlated with the magnitude of density changes. Frequencies of transferrin alleles changed the most when population density was in decline. Changes among the five phenotypic traits were generally uncorrelated with one another, except for skull and mandible shape. Nonmetric traits were selectively neutral when assessed with Q_ST/F_ST analysis, whereas mandible size, mandible shape, and skull shape showed evidence of fairly strong selection. Selection on skull size was weak or nonexistent. We discuss how different assumptions about the genetic components of variance affect Q_ST estimates when phenotypic variances are substituted for genetic ones. We also found that change in mandible size, mandible shape, skull size, and skull shape were greater than expected under a neutral model given reasonable assumptions about heritability and effective population size.     

Analysis of genotypic diversity and relationships among Pseudomonas stutzeri strains by PCR-based genomic fingerprinting and multilocus enzyme electrophoresis.

Molecular fingerprinting procedures including random amplified polymorphic DNA-PCR (RAPD), repetitive extragenic palindromic PCR (rep-PCR) with REP, ERIC, and BOX primers and multilocus enzyme electrophoresis (MLEE) were used for genotypic characterization of 16 P. stutzeri strains originally isolated from marine, waste water, clinical and soil samples. A distinct genotype of each strain and overall great genotypic diversity were found within P. stutzeri. Cluster analysis (UPGMA) of the electrophoretic patterns of all PCR-based methods used resulted in concordant grouping of 8 strains. With the other strains conflicting clustering was noticed. The variability of clustering in PCR-based analyses suggested the occurrence of chromosomal rearrangements. When RAPD-, rep-PCR and MLEE fingerprints were used in a cluster analysis of combined electrophoretic patterns, the P. stutzeri strains could be differentiated into seven distinct genotypic groups. These results supported the subdivision of the species in several genomovars and reproduced, with higher resolution, the strain grouping after 16S rRNA phylogenetic reconstruction. The combined use of several fingerprint-based genotypic analyses results in higher resolutive strain clustering by UPGMA than each of the single ones analyzed separately. Additionally, this combination of individual typings proved to be reliable of the determination of the great genotypic diversity and relationships among the P. stutzeri strains.     

Assessment of the Correlation Between Two Defining Criteria for Bidirectional Isthmic Block in the Ablation of Typical Atrial Flutter

Background

A complete, bidirectional conduction block in the cavotricuspid isthmus (CTI) represents the end-point of the typical atrial flutter ablation. We investigated the correlation between two criteria for successful ablation, one based on the atrial bipolar electrogram morphology before and after complete CTI conduction block, compared to the standard criteria of differential pacing and reversal in the right atrial depolarization sequence during coronary sinus (CS) pacing.

Method

We conducted a retrospective study in 111 patients (81 males, average age 62±10 years) who underwent an atrial flutter ablation during September 2007 - July 2009 in the Cardiology - Rehabilitation Hospital, UMF Cluj-Napoca. We assessed the presence of a bidirectional block at the end of the procedure using the standard criteria. We then analyzed the morphology of the bipolar atrial electrograms adjacent to the ablation line, before and after CTI conduction block.

Results

A change from a qRs morphology to a rSr'' morphology when pacing from the coronary sinus and from a rsr'' morphology to a QRS morphology when pacing from the low-lateral right atrium was associated with a CTI conduction block. Sensitivity (Se), specificity(Sp), positive predictive value (PPV), negative predictive value (NPV) were 96%, 89%, 99% and 67% respectively.

Conclusion

Our study suggests that the analysis of the atrial bipolar electrogram next to the ablation line before and after CTI ablation may be used as a reliable criterion to validate CTI conduction block due to its high sensitivity, specificity and positive predictive value.     

Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions

Background  

Pseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF). Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages.