Benchmarking of programs that predict the position of transmembrane segments in beta-barrel proteins

We propose a method for comparative analysis of the programs that locate transmembrane segments in proteins. On this basis, we have compiled a sample of beta-barrel protein that allows unequivocal assessment of prediction quality. Upon testing of several Internet servers, B2TMR proves the best, with B2TMPRED, HMM-B2TMR, and PROFtmb following.     

The performance of several multiple-sequence alignment programs in relation to secondary-structure features for an rRNA sequence

The performances of five global multiple-sequence alignment programs (CLUSTAL W, Divide and Conquer, Malign, PileUp, and TreeAlign) were evaluated using part of the animal mitochondrial small subunit (12S) rRNA molecule. Conserved sequence motifs derived from an alignment based on secondary structural information were used to score how well each program aligned a data set of five vertebrate and five invertebrate taxa over a range of parameter values. All of the programs could align the motifs with reasonable accuracy for at least one set of parameter conditions, although if the whole sequence was considered, similarity to the structural alignment was only 25%-34%. Use of small gap costs generally gave more accurate results, although Malign and TreeAlign generated longer alignments when gap costs were low. The programs differed in the consistency of the alignments when gap cost was varied; CLUSTAL W, Divide and Conquer, and TreeAlign were the most accurate and robust, while PileUp performed poorly as gap cost values increased, and the accuracy of Malign fluctuated. Default settings for the programs did not give the best results, and attempting to select similar parameter values in different programs did not always result in more similar alignments. Poor alignment of even well-conserved motifs can occur if these are near sites with insertions or deletions. Since there is no a priori way to determine gap costs and because such costs can vary over the gene, alignment of rRNA sequences, particularly the less well conserved regions, should be treated carefully and aided by secondary structure and conserved motifs. Some motifs are single bases and so are often invisible to alignment programs. Our tests involved the most conserved regions of the 12S rRNA gene, and alignment of less well conserved regions will be more problematical. None of the alignments we examined produced a fully resolved phylogeny for the data set, indicating that this portion of 12S rRNA is insufficient for resolution of distant evolutionary relationships.     

TNT version 1.5, including a full implementation of phylogenetic morphometrics

Version 1.5 of the computer program TNT completely integrates landmark data into phylogenetic analysis. Landmark data consist of coordinates (in two or three dimensions) for the terminal taxa; TNT reconstructs shapes for the internal nodes such that the difference between ancestor and descendant shapes for all tree branches sums up to a minimum; this sum is used as tree score. Landmark data can be analysed alone or in combination with standard characters; all the applicable commands and options in TNT can be used transparently after reading a landmark data set. The program continues implementing all the types of analyses in former versions, including discrete and continuous characters (which can now be read at any scale, and automatically rescaled by TNT). Using algorithms described in this paper, searches for landmark data can be made tens to hundreds of times faster than it was possible before (from T to 3T times faster, where T is the number of taxa), thus making phylogenetic analysis of landmarks feasible even on standard personal computers.     

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH- terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.     

H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization

The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.     

Characterization of sorCS1, an alternatively spliced receptor with completely different cytoplasmic domains that mediate different trafficking in cells

We previously isolated and sequenced murine sorCS1, a type 1 receptor containing a Vps10p-domain and a leucine-rich domain. We now show that human sorCS1 has three isoforms, sorCS1a-c, with completely different cytoplasmic tails and differential expression in tissues. The b tail shows high identity with that of murine sorCS1b, whereas the a and c tails have no reported counterparts. Like the Vps10p-domain receptor family members sortilin and sorLA, sorCS1 is synthesized as a proreceptor that is converted in late Golgi compartments by furin-mediated cleavage. Mature sorCS1 bound its own propeptide with low affinity but none of the ligands previously shown to interact with sortilin and sorLA. In transfected cells, about 10% of sorCS1a was expressed on the cell surface and proved capable of rapid endocytosis in complex with specific antibody, whereas sorCS1b presented a high cell surface expression but essentially no endocytosis, and sorCS1c was intermediate. This is an unusual example of an alternatively spliced single transmembrane receptor with completely different cytoplasmic domains that mediate different trafficking in cells.     

Kalign2: high-performance multiple alignment of protein and nucleotide sequences allowing external features

In the growing field of genomics, multiple alignment programs are confronted with ever increasing amounts of data. To address this growing issue we have dramatically improved the running time and memory requirement of Kalign, while maintaining its high alignment accuracy. Kalign version 2 also supports nucleotide alignment, and a newly introduced extension allows for external sequence annotation to be included into the alignment procedure. We demonstrate that Kalign2 is exceptionally fast and memory-efficient, permitting accurate alignment of very large numbers of sequences. The accuracy of Kalign2 compares well to the best methods in the case of protein alignments while its accuracy on nucleotide alignments is generally superior. In addition, we demonstrate the potential of using known or predicted sequence annotation to improve the alignment accuracy. Kalign2 is freely available for download from the Kalign web site (http://msa.sbc.su.se/).     

Flavonoids, including an unusual flavonoid-Mg2+ salt, from roots of Cudrania cochinchinensis

Four flavonoids with 2',4'-di-oxygenated B-rings, cochinchinol A (1), cochinchinol B (2), (2R,3R)-4',7-dihydroxy-2',5-dimethoxydihydroflavonol (3), 4',7-dihydroxy-2',5-dimethoxyflavonol (4), along with 11 known compounds, were isolated from an ethanolic extract of the roots of Cudrania cochinchinensis. Their structures were elucidated by chemical and spectroscopic methods. Cochinchinol A (1) and cochinchinol B (2) have two hitherto unprecedented flavonol salt structures in natural product chemistry. Cytotoxic activities were evaluated against several different cell lines.     

Cloning, expression, and characterization of an alternatively spliced variant of human heparanase

Heparanase is an endoglycosidase that cleaves heparan sulfate in the extracellular matrix (ECM) and hence participates in ECM degradation and remodeling. Heparanase is involved in fundamental biological processes such as cancer metastasis, angiogenesis, and inflammation. Alternative splicing in the coding region of human heparanase was not reported. Here, we report the cloning of a splice variant of human heparanase that lacks exon 5 and is missing 174 bp compared to the wild-type cDNA. Splice 5 is expressed as a 55 kDa protein compared to the 65 and 50 kDa latent and active wild-type enzyme. Splice 5 was not detected in the incubation medium of tumor cells as opposed to the wild-type latent heparanase. Splice 5 escaped proteolytic cleavage, was devoid of HS degradation activity and exhibited diffused rather than granular cellular localization.     

A survival model for unthinned loblolly pine plantations that incorporates non-planted tree competition,site quality,and incidence of fusiform rust

Future biomass yields are functionally related to the number of trees surviving at a given age. A stand level survival model was developed that incorporates competition of non-planted trees, site quality, and the incidence of fusiform rust (Cronartium quercuum [Berk.] Miyabe ex Shirai f. sp. fusiforme). The model consists of a system of two equations, one of which represents the number of surviving trees infected by fusiform rust while the other represents the number of trees not infected by fusiform rust. Data from unthinned loblolly pine (Pinus taeda L.) plantations in East Texas were used to fit and evaluate the survival model and illustrate its use. The model successfully predicted that the number of surviving loblolly pine trees decreased as the number of non-planted trees increased. The model also successfully predicted the transition of loblolly pine trees from an uninfected to an infected status by fusiform rust.     

HURP wraps microtubule ends with an additional tubulin sheet that has a novel conformation of tubulin

HURP is a newly discovered microtubule-associated protein (MAP) required for correct spindle formation both in vitro and in vivo. HURP protein is highly charged with few predicted secondary and tertiary folding domains. Here we explore the effect of HURP on pure tubulin, and describe its ability to induce a new conformation of tubulin sheets that wrap around the ends of intact microtubules, thereby forming two concentric tubes. The inner tube is a normal microtubule, while the outer one is a sheet composed of tubulin protofilaments that wind around the inner tube with a 42.5° inclination. We used cryo-electron microscopy and unidirectional surface shadowing to elucidate the structure and conformation of HURP-induced tubulin sheets and their interaction with the inner microtubule. These studies clarified that HURP-induced sheets are composed of anti-parallel protofilaments exhibiting P2 symmetry. HURP is a unique MAP that not only stabilizes and bundles microtubules, but also polymerizes free tubulin into a new configuration.     

IL-20, an anti-angiogenic cytokine that inhibits COX-2 expression

COX-2 overexpression and subsequent PGE(2) production are frequently associated with non-small cell lung cancer and are implicated in tumor-mediated angiogenesis. Here, we report for the first time that IL-20 downregulates COX-2 and PGE(2) in human bronchial epithelial and endothelial cells. Flow cytometry analysis suggests that IL-20-dependent inhibition of COX-2/PGE(2) occurs through the IL-22R1/IL-20R2 dimers. In addition, we report that IL-20 exerts anti-angiogenic effects, inhibiting experimental angiogenesis. IL-20-mediated inhibition of PMA-induced angiogenesis occurs through the COX-2 regulatory pathway. Altogether our findings revealed that IL-20 is a negative modulator of COX-2/PGE(2) and inhibits angiogenesis.     

Sucrose may play an additional role to that of an osmolyte in Synechocystis sp. PCC 6803 salt-shocked cells.

The role of sucrose in cyanobacteria is still not fully understood. It is generally considered a salt-response molecule, and particularly, in Synechocystis sp. strain PCC 6803, it is referred as a secondary osmolyte. We showed that sucrose accumulates transiently in Synechocystis cells at early stages of a salt shock, which could be ascribed to salt activation of sucrose-phosphate synthase (SPS, UDP-glucose: D-fructose-6-phosphate 2-alpha-D-glucosyltransferase; EC 2.4.1.14), the key enzyme in sucrose synthesis pathway, and to an increase of the expression of the SPS encoding gene. Experiments with a mutant strain impaired in sucrose biosynthesis showed that sucrose is essential in stationary phase cells to overcome a later salt stress. Taken together, these results led us to suggest a more intricate function for sucrose than to be an osmoprotectant compound.     

Some features of imaging of the processes occurring in an extended arc discharge in atmospheric-pressure air

Processes occurring in the low-temperature plasma of extended quasi-stationary arc discharges in air between graphite electrodes are investigated. Along with the conventional (constricted) discharge geometry, other discharge modes—diffuse (distributed) and diffuse-constricted—are studied. Contraction, stratification, and shunting processes are considered. Current oscillation modes are revealed that are caused by the interaction between the cathode and anode jets and the origination of plasma jets and solid particles from the locally overheated anode surface. 1 The use of graphite electrodes with standard atmospheric pressure excludes the presence of the liquid phase in the electrode spots