Multi‐proxy analyses of Late Cretaceous coprolites from Germany

A total of 462 coprolites from three localities exposing Upper Cretaceous deposits in the Münster Basin, northwestern Germany, have been subjected to an array of analytical techniques, with the aim of elucidating ancient trophic structures and predator–prey interactions. The phosphatic composition, frequent bone inclusions, size and morphology collectively suggest that most, if not all, coprolites were produced by carnivorous (predatory or scavenging) vertebrates. The bone inclusions further indicate that the coprolite producers preyed principally upon fish. Putative host animals include bony fish, sharks and marine reptiles – all of which have been previously recorded from the Münster Basin. The presence of borings and other traces on several coprolites implies handling by coprophagous organisms. Remains of epibionts are also common, most of which have been identified as the encrusting bivalve Atreta. Palynological analyses of both the coprolites and host rocks reveal a sparse assemblage dominated by typical Late Cretaceous dinoflagellates, and with sub‐ordinate fern spores, conifer pollen grains and angiosperm pollen grains. The dinoflagellate key taxon Exochosphaeridium cenomaniense corroborates a Cenomanian age for the Plenus Marl, from which most studied coprolites derive. The findings of this study highlight the potential of a multi‐proxy approach when it comes to unravelling the origin, composition and importance of coprolites in palaeoecosystem analyses.     

Low affinity, antibody binding of an Escherichia coli-derived component

Abstract This investigation describes the detection of a component in Escherichia coli capable of binding a large proportion of human antibody variable domains including otherwise highly monospecific antibodies induced by an in vivo antibody response. This interaction is of low affinity, but cross-linking of IgG molecules by e.g. anti-immunoglobulin preparations, provides a sufficient degree of multivalency to promote a high avidity interaction. This binding which occurs both with κ and λ light chain-containing antibodies, appears to involve the variable region of human antibodies making it a superantigen-like activity. This is proposed based on the facts that: (i) different human antibodies of IgG1 isotype appear to bind to different extents suggesting that variable domain differences determine the binding activity; and (ii) addition of soluble antigen abrogates the interaction with the E. coli -derived molecule. Future studies of the nature and possible in vivo consequences of these interactions are warranted since any superantigen activity associated with this binding might affect the human immune response occurring as a consequence of E. coli infections.     

Matrilin-2, a large, oligomeric matrix protein, is expressed by a great variety of cells and forms fibrillar networks

Matrilin-2 is a member of the protein superfamily with von Willebrand factor type A-like modules. Mouse matrilin-2 cDNA fragments were expressed in 293-EBNA cells, and the protein was purified, characterized, and used to immunize rabbits. The affinity-purified antiserum detects matrilin-2 in dense and loose connective tissue structures, subepithelial connective tissue of the skin and digestive tract, specialized cartilages, and blood vessel walls. In situ hybridization of 35S-labeled riboprobes localizes the matrilin-2 mRNA to fibroblasts of dermis, tendon, ligaments, perichondrium, and periosteum; connective tissue elements in the heart; smooth muscle cells; and epithelia and loose connective tissue cells of the alimentary canal and respiratory tract. RNA blot hybridization and immunoblotting revealed both matrilin-2 mRNA and protein in cultures of a variety of cell types, confirming the tissue distribution. Alternative splicing affects a module unique for matrilin-2 in all of the above RNA sources. SDS-polyacrylamide gel electrophoresis and electron microscopy reveals matrilin-2 from tissue extracts and cell line cultures as a mixture of mono-, di-, tri-, and tetramers. Matrilin-2 is substituted with N-linked oligosaccharides but not with glycosaminoglycans. Because of other, yet unidentified, cell-type dependent posttranslational modifications, the monomer is heterogeneous in size. Immunofluorescence showed that matrilin-2 functions by forming an extracellular, filamentous network.     

Matrilin-3 is dispensable for mouse skeletal growth and development

Matrilin-3 belongs to the matrilin family of extracellular matrix (ECM) proteins and is primarily expressed in cartilage. Mutations in the gene encoding human matrilin-3 (MATN-3) lead to autosomal dominant skeletal disorders, such as multiple epiphyseal dysplasia (MED), which is characterized by short stature and early-onset osteoarthritis, and bilateral hereditary microepiphyseal dysplasia, a variant form of MED characterized by pain in the hip and knee joints. To assess the function of matrilin-3 during skeletal development, we have generated Matn-3 null mice. Homozygous mutant mice appear normal, are fertile, and show no obvious skeletal malformations. Histological and ultrastructural analyses reveal endochondral bone formation indistinguishable from that of wild-type animals. Northern blot, immunohistochemical, and biochemical analyses indicated no compensatory upregulation of any other member of the matrilin family. Altogether, our findings suggest functional redundancy among matrilins and demonstrate that the phenotypes of MED disorders are not caused by the absence of matrilin-3 in cartilage ECM.     

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.     

A novel mammalian display system for the selection of protein-protein interactions by decoy receptor engagement

The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component.     

Process considerations and economic evaluation of two-step steam pretreatment for production of fuel ethanol from softwood

To increase the overall ethanol yield from softwood, the steam pretreatment stage can be carried out in two steps. The two-step pretreatment process was evaluated from a techno-economic standpoint and compared with the one-step pretreatment process. The production plants considered were designed to utilize spruce as raw material and have a capacity of 200,000 tons/year. The two-step process resulted in a higher ethanol yield and a lower requirement for enzymes. However, the two-step process is more capital-intensive and has a higher energy requirement. The estimated ethanol production cost was the same, 4.13 SEK/L (55.1 cent /L) for both alternatives. For the two-step process different energy-saving options were considered, such as a higher concentration of water-insoluble solids in the filter cake before the second step, and the possibility of excluding the pressure reduction between the steps. The most optimistic configuration, with 50% water-insoluble solids in the filter cake in the feed to the second pretreatment step, no pressure reduction between the pretreatment steps, and 77% overall ethanol yield (0.25 kg EtOH/kg dry wood), resulted in a production cost of 3.90 SEK/L (52.0 cent /L). This shows the potential for the two-step pretreatment process, which, however, remains to be verified in pilot trials.     

Size and structure characterization of ethylhydroxyethyl cellulose by the combination of field-flow fractionation with other techniques. Investigation of ultralarge components

Ethylhydroxyethyl cellulose (EHEC) of three different viscosity classes (EHEC I, II, and III) was analyzed by programmed cross-flow asymmetrical flow field-flow fractionation coupled to multiangle light scattering and refractive index detectors to determine their size and molar mass distribution. Two size populations were detected in the two lower viscosity classes, EHEC I and II, one high molar mass and one ultrahigh molar mass (UHM). The two covered molar masses from 10(4) up to 10(9) g X mol(-1). The highest viscosity class EHEC III was less size-dispersed covering molar masses from 5 x 10(5) to 5 x 10(7) g.mol(-1). Filtering of the EHEC II solution removed small amounts of compact UHM material. Enzyme treatments were performed on EHEC II to further characterize it. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and anion ion-exchange chromatography coupled to pulsed amperometric detection showed that the UHM component contained EHEC.