BuildBeta—A system for automatically constructing beta sheets

We describe a method that can thoroughly sample a protein conformational space given the protein primary sequence of amino acids and secondary structure predictions. Specifically, we target proteins with β‐sheets because they are particularly challenging for ab initio protein structure prediction because of the complexity of sampling long‐range strand pairings. Using some basic packing principles, inverse kinematics (IK), and β‐pairing scores, this method creates all possible β‐sheet arrangements including those that have the correct packing of β‐strands. It uses the IK algorithms of ProteinShop to move α‐helices and β‐strands as rigid bodies by rotating the dihedral angles in the coil regions. Our results show that our approach produces structures that are within 4–6 Å RMSD of the native one regardless of the protein size and β‐sheet topology although this number may increase if the protein has long loops or complex α‐helical regions. Proteins 2010. © Published 2009 Wiley‐Liss, Inc.     

Interferon-gamma synergises with tumour necrosis factor and lymphotoxin-alpha to enhance the mRNA and protein expression of adhesion molecules in mouse brain endothelial cells

Changes to the cerebral microvasculature are evident during cerebral malaria (CM). Activation of the endothelium is likely to be due to the actions of cytokines, circulating levels of which are elevated during CM. Endothelial cells are known to up-regulate the expression of cellular adhesion molecules, which can lead to cellular sequestration and obstruction of vessels. However, it is unknown whether cytokines synergise in the up-regulation of the adhesion molecules involved in CM. In this study, the mRNA and/or protein expression of the adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin and E-Selectin were examined in a mouse brain endothelial cell line. Endothelial cells were stimulated with interferon-gamma (IFN-gamma), tumour necrosis factor (TNF) and lymphotoxin-alpha (LT-alpha), alone or in combination. The expression of ICAM-1, VCAM-1, P-selectin and E-Selectin mRNA in mouse brain endothelial cells by TNF and/or LT-alpha was found to be significantly enhanced in the presence of IFN-gamma. The same synergistic effect was found when analyzing ICAM-1 protein expression in cytokine stimulated mouse brain endothelial cells. The findings show that cytokines can synergise to influence gene expression and protein expression in a mouse brain endothelial cell line.     

Cerato-platanin protein is located in the cell walls of ascospores, conidia and hyphae of Ceratocystis fimbriata f. sp. platani

Cerato-platanin (CP), a protein of about 12.4 kDa from Ceratocystis fimbriata f. sp. platani (Cfp), accumulated in the mycelium and was located in the cell walls of Cfp ascospores, hyphae and conidia suggesting that this protein had a role in forming the fungal cell wall apart from the already known fact that it is secreted early in culture and elicits phytoalexin synthesis and/or plant cell death. The finding was obtained with three immunological techniques: a quantitative ELISA which determines the amount of CP in the mycelium, an immunofluorescence assay, and immunogold labelling to define the exact localization of CP in the Cfp cells.     

Efficient Bayesian Sample Size Calculation for Designing a Clinical Trial with Multi‐Cluster Outcome Data

Health care utilization and outcome studies call for hierarchical approaches. The objectives were to predict major complications following percutaneous coronary interventions by health providers, and to compare Bayesian and non‐Bayesian sample size calculation methods. The hierarchical data structure consisted of: (1) Strata: PGY4, PGY7, and physician assistant as providers with varied experiences; (2) Clusters: k_s providers per stratum; (3) Individuals: n_s patients reviewed by each provider. The main outcome event illustrated was mortality modeled by a Bayesian beta‐binomial model. Pilot information and assumptions were utilized to elicit beta prior distributions. Sample size calculations were based on the approximated average length, fixed at 1%, of 95% posterior intervals of the mean event rate parameter. Necessary sample sizes by both non‐Bayesian and Bayesian methods were compared. We demonstrated that the developed Bayesian methods can be efficient and may require fewer subjects to satisfy the same length criterion.     

Molecular Characterization of a cDNA Encoding Functional Human CLK4 Kinase and Localization to Chromosome 4q35

Phosphorylated serine- and arginine-rich (SR) proteins play an important role in the formation of spliceosomes, possibly controlling the regulation of alternative splicing. Enzymes that phosphorylate the SR proteins belong to the family of CDC2/CDC28-like kinases (CLK). Employing nucleotide sequence comparison of human expressed sequence tag sequences to the murine counterpart, we identified, cloned, and recombinantly expressed the human orthologue to the murine CLK4 cDNA. When fused to glutathione S-transferase, the catalytically active human CLK4 is able to autophosphorylate and to phosphorylate myelin basic protein, but not histone H2B as a substrate. Inspection of mRNA accumulation demonstrated gene expression in all human tissues, with the most prominent abundance in liver, kidney, brain, and heart. Using fluorescence in situ hybridization, the human CLK4 cDNA was localized to band q35 on chromosome 4.     

Enrichment strategies for siRNA-transfected cells in an untransfected background

Gene silencing experiments in difficult-to-transfect cells are often hampered by the presence of a background of untransfected cells. We present proof-of-concept data from two different strategies for enrichment of siRNA-transfected cells. In the first approach, a heterologous surface antigen is expressed from a plasmid that is co-transfected with an siRNA targeting an endogenous mRNA. The surface antigen is then used for enrichment of successfully transfected cells using antibody-coated magnetic particles. In the second strategy, a eukaryotic antibiotic resistance gene is expressed from a co-transfected plasmid. Addition of the corresponding antibiotic 24h after transfection results in killing of untransfected cells, which can be washed away. Elimination of untransfected cells will allow more accurate interpretation of the effects of gene silencing.     

Knocking down the diencephalic thyrotropin-releasing hormone precursor gene normalizes obesity-induced hypertension in the rat

We recently showed that diencephalic TRH may mediate the central leptin-induced pressor effect. Here, to study the role of TRH in obesity-induced hypertension (OIH), we used a model of OIH produced by a high-fat diet (HFD, 45 days) in male Wistar rats. After 4 wk, body weight and systolic arterial blood pressure (SABP) increased in HFD animals. Plasma leptin was correlated with peritoneal adipose tissue. Then, we treated OIH animals with an antisense oligodeoxynucleotide and small interfering (si)RNA against the prepro-TRH. Antisense significantly decreased diencephalic TRH content and SABP at 24 and 48 h posttreatment. Similar effects were observed with siRNA against prepro-TRH but for up to 4 wk. Conversely, vehicle, an inverted antisense sequence and siRNA against green fluorescence protein, produced no changes. SABP decrease seems to be owing to an inhibition of the obesity-enhanced sympathetic outflow but not to an alteration in thyroid status. Using a simple OIH model we demonstrated, for the first time, that central TRH participates in the hypertension induced by body weight gain probably through its well-known action on sympathetic activity. Thus the TRH-leptin interaction may contribute to the strong association between hypertension and obesity.     

The insertion of the antimicrobial peptide dicynthaurin monomer in model membranes: thermodynamics and structural characterization

This paper is focused on the thermodynamics and the structural investigation of the interaction of the antimicrobial peptide dicynthaurin monomer with model lipid membranes composed of mixtures of 1-palmitoyl-2-oleyl-glycerophosphocholine and -glycerophosphoglycerol. The thermodynamic binding parameters as obtained by isothermal titration calorimetry reveal strong binding toward the lipid model system dominated by large chemical binding constants which exceeds the electrostatic binding effects and thus suggests insertion of the amphipathic alpha-helical peptide into the hydrophobic membrane core. Circular dichroism study shows that the peptide exhibits trans-membrane alpha-helix secondary structure. Neutron diffraction measurements using partially deuterated sequences were successfully applied to determine the orientation of the peptide thus proving insertion into the hydrophobic membrane core. This insertion and the formation of higher order porelike aggregates is assumed to be the most relevant event in microbial membrane perturbation that in vivo finally leads to bacterial cell death on a fast time scale.