Sec-mediated transport of posttranslationally dehydrated peptides in Lactococcus lactis

Nisin is a lanthionine-containing antimicrobial peptide produced by Lactococcus lactis. Its (methyl)lanthionines are introduced by two posttranslational enzymatic steps involving the dehydratase NisB, which dehydrates serine and threonine residues, and the cyclase NisC, which couples these dehydrated residues to cysteines, yielding thioether-bridged amino acids called lanthionines. The prenisin is subsequently exported by the ABC transporter NisT and extracellularly processed by the peptidase NisP. L. lactis expressing the nisBTC genes can modify and secrete a wide range of nonlantibiotic peptides. Here we demonstrate that in the absence of NisT and NisC, the Sec pathway of L. lactis can be exploited for the secretion of dehydrated variants of therapeutic peptides. Furthermore, posttranslational modifications by NisB and NisC still occur even when the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively. However, transport of fully modified prenisin via the Sec pathway is impaired. The extent of NisB-mediated dehydration could be improved by raising the intracellular concentration NisB or by modulating the export efficiency through altering the signal sequence. These data demonstrate that besides the traditional lantibiotic transporter NisT, the Sec pathway with an established broad substrate range can be utilized for the improved export of lantibiotic enzyme-modified (poly)peptides.     

Nano-cluster composite structure of calcitic sponge spicules--a case study of basic characteristics of biominerals

Spicules of calcareous sponges are elaborately shaped skeletal elements that nonetheless show characteristics of calcite single-crystals. Our atomic force microscopic and transmission electron microscopic investigation of the triradiate spicules of the sponge Pericharax heteroraphis reveals a nano-cluster structure with mostly well-aligned small crystal domains and pockets with accumulated domain misalignments. Combined high-resolution and energy-filtering transmission electron microscopy revealed carbon enrichments located in between crystal domain boundaries, which strongly suggests an intercalated network-like proteinaceous organic matrix. This matrix is proposed to be involved in the nano-clustered calcite precipitation via a transient phase that may enable a 'brick-by-brick' formation of composite and yet single-crystalline spicules with elaborate morphologies. This composite cluster structure reduces the brittleness of the material by dissipating strain energy and deflecting crack propagation from the calcite cleavage planes, but the lattice symmetry and anisotropic growth properties of calcite still play a major role in the morphogenesis of these unusual calcite single-crystals. Our structural, crystallographic, textural, and chemical analysis of sponge spicules corroborates the view that nano-clustered crystal growth, induced by organic matrices, is a basic characteristic of biomineralisation that enables the production of composite materials with elaborate morphologies.     

More robust detection of motifs in coexpressed genes by using phylogenetic information

Background  

Several motif detection algorithms have been developed to discover overrepresented motifs in sets of coexpressed genes. However, in a noisy gene list, the number of genes containing the motif versus the number lacking the motif might not be sufficiently high to allow detection by classical motif detection tools. To still recover motifs which are not significantly enriched but still present, we developed a procedure in which we use phylogenetic footprinting to first delineate all potential motifs in each gene. Then we mutually compare all detected motifs and identify the ones that are shared by at least a few genes in the data set as potential candidates.     

Diffusion limitation: a possible source for the occurrence of doughnut patterns on DNA microarrays

Doughnut shaped hybridization patterns on DNA microarrays are mainly allocated to spotting or drying artifacts. The present study reports on results obtained from four different approaches that when combined generate a better view on the occurrence of these patterns. This study points out that doughnuts are not only formed during the spotting and drying process, but the hybridization process itself can be considered as an important cause. A combination of computer simulations, theoretical, optical, and experimental techniques shows how ring-shaped hybridization patterns occur when diffusion-limited conditions are present during the hybridization process. The theoretical assumptions as well as the simulations indicate that, for the basic geometry of a microarray hybridization experiment, a large amount of binding molecules reach the spot from the sides (and not from above the spot), leading to a preferential binding on the rims of the spot. These patterns seem to occur especially during hybridization with short oligonucleotides that have a very high binding probability and fast hybridization kinetics. Longer target DNA molecules lead to a more evenly distributed intensity signal. Furthermore, the diffusion-limited conditions also lead to pronounced hybridization intensity patterns on the scale of a whole spot block, where larger intensities are obtained on the edges of the block compared with the spots laying in the center of the block.     

A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation

We describe the tripeptide neutrophil chemoattractant N-acetyl Pro-Gly-Pro (PGP), derived from the breakdown of extracellular matrix (ECM), which shares sequence and structural homology with an important domain on alpha chemokines. PGP caused chemotaxis and production of superoxide through CXC receptors, and administration of peptide caused recruitment of neutrophils (PMNs) into lungs of control, but not CXCR2-deficient mice. PGP was generated in mouse lung after exposure to lipopolysaccharide, and in vivo and in vitro blockade of PGP with monoclonal antibody suppressed PMN responses as much as chemokine-specific monoclonal antibody. Extended PGP treatment caused alveolar enlargement and right ventricular hypertrophy in mice. PGP was detectable in substantial concentrations in a majority of bronchoalveolar lavage samples from individuals with chronic obstructive pulmonary disease, but not control individuals. Thus, PGP's activity links degradation of ECM with neutrophil recruitment in airway inflammation, and PGP may be a biomarker and therapeutic target for neutrophilic inflammatory diseases.     

Proteome profiling in the rat Harderian gland

The Harderian gland is an orbital gland located behind the ocular bulb in most terrestrial vertebrates probably functioning for production of lipid secretion to protect the eye. We herein present a protein reference database of the rat Harderian gland that may serve as analytical tool for future proteomic work, report lipid and porphyrin handling cascades, address sequence conflicts and report structures that have not been so far described by proteomics methods.     

Traditional biomolecular structure determination by NMR spectroscopy allows for major errors

One of the major goals of structural genomics projects is to determine the three-dimensional structure of representative members of as many different fold families as possible. Comparative modeling is expected to fill the remaining gaps by providing structural models of homologs of the experimentally determined proteins. However, for such an approach to be successful it is essential that the quality of the experimentally determined structures is adequate. In an attempt to build a homology model for the protein dynein light chain 2A (DLC2A) we found two potential templates, both experimentally determined nuclear magnetic resonance (NMR) structures originating from structural genomics efforts. Despite their high sequence identity (96%), the folds of the two structures are markedly different. This urged us to perform in-depth analyses of both structure ensembles and the deposited experimental data, the results of which clearly identify one of the two models as largely incorrect. Next, we analyzed the quality of a large set of recent NMR-derived structure ensembles originating from both structural genomics projects and individual structure determination groups. Unfortunately, a visual inspection of structures exhibiting lower quality scores than DLC2A reveals that the seriously flawed DLC2A structure is not an isolated incident. Overall, our results illustrate that the quality of NMR structures cannot be reliably evaluated using only traditional experimental input data and overall quality indicators as a reference and clearly demonstrate the urgent need for a tight integration of more sophisticated structure validation tools in NMR structure determination projects. In contrast to common methodologies where structures are typically evaluated as a whole, such tools should preferentially operate on a per-residue basis.     

PlantMetabolomics.org: A Web Portal for Plant Metabolomics Experiments

PlantMetabolomics.org (PM) is a web portal and database for exploring, visualizing, and downloading plant metabolomics data. Widespread public access to well-annotated metabolomics datasets is essential for establishing metabolomics as a functional genomics tool. PM integrates metabolomics data generated from different analytical platforms from multiple laboratories along with the key visualization tools such as ratio and error plots. Visualization tools can quickly show how one condition compares to another and which analytical platforms show the largest changes. The database tries to capture a complete annotation of the experiment metadata along with the metabolite abundance databased on the evolving Metabolomics Standards Initiative. PM can be used as a platform for deriving hypotheses by enabling metabolomic comparisons between genetically unique Arabidopsis (Arabidopsis thaliana) populations subjected to different environmental conditions. Each metabolite is linked to relevant experimental data and information from various annotation databases. The portal also provides detailed protocols and tutorials on conducting plant metabolomics experiments to promote metabolomics in the community. PM currently houses Arabidopsis metabolomics data generated by a consortium of laboratories utilizing metabolomics to help elucidate the functions of uncharacterized genes. PM is publicly available at http://www.plantmetabolomics.org.In the post genomics era, metabolomics is fast emerging as a vital source of information to aid in solving systems biology puzzles with an emphasis on metabolic solutions. Metabolomics is the science of measuring the pool sizes of metabolites (small molecules of M_r ≤ 1,000 D), which collectively define the metabolome of a biological sample (Fiehn et al., 2000; Hall et al., 2002). Coverage of the entire plant metabolome is a daunting task as it is estimated that there are over 200,000 different metabolites within the plant kingdom (Goodacre et al., 2004). Although technology is rapidly advancing, there are still large gaps in our knowledge of the plant metabolome.Despite this lack of complete knowledge and the immense metabolic diversity among plants, metabolomics has become a key analytical tool in the plant community (Hall et al., 2002). This has led to the emergence of multiple experimental and analytical platforms that collectively generate millions of metabolite data points. Because of this vast amount of data, the development of public databases to capture information from metabolomics experiments is vital to provide the scientific community with comprehensive knowledge about metabolite data generation, annotation, and integration with metabolic pathway data. Some examples of these public databases are given below. The Human Metabolome Project contains comprehensive data for more than 2,000 metabolites found within the human body (Wishart et al., 2007). The Golm Database is a repository that provides access to mass spectrometry (MS) libraries, metabolite profiling experiments, and related information from gas chromatography (GC)-MS experimental platforms, along with tools to integrate this information with other systems biology knowledge (Kopka et al., 2005). The Madison Metabolomics Consortium Database contains primarily NMR spectra for Arabidopsis (Arabidopsis thaliana) and features thorough NMR search tools (Cui et al., 2008). SetupX and Binbase provide a framework that combines MS data and biological metadata for steering laboratory work flows and employs automated metabolite annotation (Scholz and Fiehn, 2007).A single analytical technique cannot identify and quantify all the metabolites found in plants. Thus, PlantMetabolomics.org (PM) was developed to provide a portal for accessing publicly available MS-based plant metabolomics experimental results from multiple analytical and separation techniques. PM also follows the emerging metabolomics standards for experiment annotation. PM has extensive annotation links between the identified metabolites and metabolic pathways in AraCyc (Mueller et al., 2003) at The Arabidopsis Information Resource (Rhee et al., 2003) and the Plant Metabolic Network (www.plantcyc.org), the Kyoto Encyclopedia of Genes and Genomes (KEGG; Kanehisa et al., 2004), and MetNetDB (Wurtele et al., 2007).Standards for the annotation of metabolomics experiments are still under active development and the metadata types collected in PM are based on the recommendations of the Metabolomics Standards Initiative (MSI; Fiehn et al., 2007a) and the Minimal Information for a Metabolomic Experiment (Bino et al., 2004) standards. MSI attempts to capture the complete annotation of metabolomics experiments and includes metadata of the experiments along with the metabolite abundance data. The initial database schema design was guided by the schema proposed in the Architecture for Metabolomics project (Jenkins et al., 2004).     

PHOTOPHYSIOLOGY IN TWO SOUTHERN OCEAN PHYTOPLANKTON TAXA: PHOTOSYNTHESIS OF PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE) AND FRAGILARIOPSIS CYLINDRUS (BACILLARIOPHYCEAE) UNDER SIMULATED MIXED‐LAYER IRRADIANCE1

In the Ross Sea, the prymnesiophyte Phaeocystis antarctica G. Karst. dominates deeply mixed water columns, while diatoms dominate shallower mixed layers. Understanding what controls the dynamics of these two phytoplankton taxa is essential because they dominate virtually all coastal polar waters, have different nutrient utilization characteristics, and support dissimilar food webs. We cultured two strains of P. antarctica and one strain of the diatom Fragilariopsis cylindrus (Grunow) Willi Krieg under three dynamic irradiance regimes that simulated different mixed‐layer depths and measured their photosynthetic characteristics, cellular pigment concentrations, and cellular carbon and nitrogen content. In both species, chl a–normalized maximum carbon uptake rate (P_m^* ) and specific growth rate were highest in the deeply mixed treatment that had a dark period. In all irradiance treatments, both (P_m^* ) and photosynthetic efficiency (α*) were greater for the two P. antarctica strains than for the F. cylindrus strain. In contrast, P. antarctica strains were more susceptible to photoinhibition (β*) than the F. cylindrus strain. When photosynthetic rates of each phytoplankton taxon were normalized by cellular particulate organic carbon (POC), the difference in the maximal photosynthetic rate () was generally reduced. In the dynamic irradiance treatment that simulated the shallowest mixed‐layer irradiance, all three phytoplankton had similar ; however, the diatom had a 2‐fold higher POC‐normalized photosynthetic efficiency (α^C). Finally, we performed calculations using the measured POC‐normalized photosynthetic parameters to show that α^C and can play a greater role than β^C in determining the competitive outcome between P. antarctica and F. cylindrus in both shallow and deep mixed‐layer environments of the Ross Sea.     

Two <Emphasis Type="Italic">Pione</Emphasis> species (Hadromerida,Clionaidae) from the Red Sea: a taxonomical challenge

Boring sponges of the genus Pione (Hadromerida, Clionaidae) are easily recognizable due to their spiculation. However, species identification is challenging, as the potentially diagnostic morphological character states of different species often overlap. For this reason, this group of species is frequently referred to as the ‘Pione vastifica complex’, after the most well-studied species of the genus. Boring-sponge samples were collected in the Red Sea and identified as P. cf. lampa and P. cf. vastifica, respectively. So far, these two species names have usually been considered as valid, although some authors suggested them to be synonymous. Morphological analyses were performed on spicules and micro-erosion patterns by means of both light and scanning electron microscopy. Two apparent morphotypes can be distinguished, mainly by the growth form, but statistical analysis does not support a clear separation in two species. In addition, a DNA barcoding approach using sequences of CO1 has not identified any nucleotide sequence differences. These data support the hypothesis that P. cf. lampa and P. cf. vastifica from the Red Sea are conspecific.