Effect of salinity on composition, viability and germination of seeds of Chenopodium quinoa Willd

Salinity influences plant growth, seed yield and seed quality even of halophytic crops such as Chenopodium quinoa. Plant growth, total seed yield, number of seeds, fresh weight and dry weight of seeds, were all significantly reduced in the presence of salinity. Only at high salinity did the content of proteins (as well as total N) increase significantly in the seeds whereas the content of total carbohydrates (as well as total C) decrease. Aside from that the capacity for germination was diminished by a reduced seed size and a disproportionate reduction of the volume of the perisperm. However, the reduced capacity seemed to be compensated by an accelerated germination owing to high Na and Cl concentrations leading to a low water potential in the walls of the plant ovary. At high salinity the passage of NaCl to the seed interior was hindered by the seed cover. There was an obvious gradient between potentially toxic (Na and Cl) and essentially needed elements (K, Mg, Ca, P and S) across the seed coat of salt treated plants and also a significant change of the distribution of elements in the embryo. The results indicate a highly protected seed interior leading to a high salinity resistance of quinoa seeds.     

Exon discovery by genomic sequence alignment

MOTIVATION: During evolution, functional regions in genomic sequences tend to be more highly conserved than randomly mutating 'junk DNA' so local sequence similarity often indicates biological functionality. This fact can be used to identify functional elements in large eukaryotic DNA sequences by cross-species sequence comparison. In recent years, several gene-prediction methods have been proposed that work by comparing anonymous genomic sequences, for example from human and mouse. The main advantage of these methods is that they are based on simple and generally applicable measures of (local) sequence similarity; unlike standard gene-finding approaches they do not depend on species-specific training data or on the presence of cognate genes in data bases. As all comparative sequence-analysis methods, the new comparative gene-finding approaches critically rely on the quality of the underlying sequence alignments. RESULTS: Herein, we describe a new implementation of the sequence-alignment program DIALIGN that has been developed for alignment of large genomic sequences. We compare our method to the alignment programs PipMaker, WABA and BLAST and we show that local similarities identified by these programs are highly correlated to protein-coding regions. In our test runs, PipMaker was the most sensitive method while DIALIGN was most specific. AVAILABILITY: The program is downloadable from the DIALIGN home page at http://bibiserv.techfak.uni-bielefeld.de/dialign/.     

Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2

The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.     

Separation of sequences from host-pathogen interface using triplet nucleotide frequencies

The identification of genes involved in host-pathogen interactions is important for the elucidation of mechanisms of disease resistance and host susceptibility. A traditional way to classify the origin of genes sampled from a pool of mixed cDNA is through sequence similarity to known genes from either the pathogen or host organism or other closely related species. This approach does not work when the identified sequence has no close homologues in the sequence databases. In our previous studies, we classified genes using their codon frequencies. This method, however, explicitly required the prediction of CDS regions and thus could not be applied to sequences composed from the non-coding regions of genes. In this study, we show that the use of sliding-window triplet frequencies extends the application of the algorithm to both coding and non-coding sequences and also increases the prediction accuracy of a Support Vector Machine classifier from 95.6+/-0.3 to 96.5+/-0.2. Thus the use of the triplet frequencies increased the prediction accuracy of the new method by more than 20% compared to our previous approach. A functional analysis of sequences detected gene families having significantly higher or lower probability to be correctly classified compared to the average accuracy of the method is described. The server to perform classification of EST sequences using triplet frequencies is available at (URL: http://mips.gsf.de/proj/est3).     

Super paramagnetic clustering of protein sequences

Background  

Detection of sequence homologues represents a challenging task that is important for the discovery of protein families and the reliable application of automatic annotation methods. The presence of domains in protein families of diverse function, inhomogeneity and different sizes of protein families create considerable difficulties for the application of published clustering methods.     

On the competition for available zinc

Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.     

Insecticidal anthranilic diamides: a new class of potent ryanodine receptor activators

A novel class of anthranilic diamides has been discovered with exceptional insecticidal activity on a range of Lepidoptera. These compounds have been found to exhibit their action by release of intracellular Ca2+ stores mediated by the ryanodine receptor. The discovery, synthesis, structure-activity, and biological results are presented.     

Role of Integrins and Evidence for two Distinct Mechanisms Mediating Human Colorectal Carcinoma Cell Interaction with Peritoneal Mesothelial Cells and Extracellular Matrix

Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Col 15 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-β1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell—mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.