Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions

Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg α-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel β-sheet complementation as observed for the clumping factor A·γ-chain peptide complex. The β-sheet complementation is shorter in the ClfB·Fg α-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.     

ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice

Reducing the cost of vaccine production is a key priority for veterinary research, and the possibility of heterologously expressing antigen in plants provides a particularly attractive means of achieving this. Here, we report the expression of the avian influenza virus haemagglutinin (AIV HA) in tobacco, both as a monomer and as a trimer in its native and its ELPylated form. We firstly presented evidence to produce stabilized trimers of soluble HA in plants. ELPylation of these trimers does not influence the trimerization. Strong expression enhancement in planta caused by ELPylation was demonstrated for trimerized H5‐ELP. ELPylated trimers could be purified by a membrane‐based inverse transition cycling procedure with the potential of successful scale‐up. The trimeric form of AIV HA was found to enhance the HA‐specific immune response compared with the monomeric form. Plant‐derived AIV HA trimers elicited potentially neutralizing antibodies interacting with both homologous virus‐like particles from plants and heterologous inactivated AIV. ELPylation did not influence the functionality and the antigenicity of the stabilized H5 trimers. These data allow further developments including scale‐up of production, purification and virus challenge experiments with the final goal to achieve suitable technologies for efficient avian flu vaccine production.     

Synthesis of active oryzacystatin I in transgenic potato plants

Summary Transformation of potato (Solanum tuberosum L.) with cysteine proteinase inhibitor (PI) genes represents a potential way of controlling the major insect pest Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The present study describes the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an oryzacystatin I (OCI) cDNA clone linked to a CaMV 35S promoter. The transgenic plants accumulated active OCI in potato leaves, as demonstrated by the papain-inhibitory activity of transgenic plant leaf extracts. In addition to their anti-papain activity, the extracts also caused a partial but significant inhibition of CPB digestive proteinases, similar to that observed with pure inhibitors. Recombinant OCI did not alter the activity of the major potato leaf endogenous proteinases, which seemed to be of the serine-type. Therefore we suggest that the OCI cDNA can be used for the production of CPB-resistant transgenic potato plants without interfering with endogenous proteinases of these plants.Abbreviations CPB Colorado potato beetle - E-64 trans-epoxy-succinyl-L-leucylamido (4-guanidino) butane - OCI oryzacystatin I - PI proteinase inhibitor - PMSF phenylmethylsulfonyl fluoride     

Suppressive subtractive hybridization of and differences in gene expression content of calcifying and noncalcifying cultures of Emiliania huxleyi strain 1516

The marine coccolithophorid Emiliania huxleyi is a cosmopolitan alga intensely studied in relation to global carbon cycling, biogeochemistry, marine ecology, and biomineralization processes. The biomineralization capabilities of coccolithophorids have attracted the attention of scientists interested in exploiting this ability for the development of materials science and biomedical and biotechnological applications. Although it has been well documented that biomineralization in E. huxleyi is promoted by growth under phosphate-limited conditions, the genes and proteins that govern the processes of calcification and coccolithogenesis remain unknown. Suppressive subtractive hybridization (SSH) libraries were constructed from cultures grown in phosphate-limited and phosphate-replete media as tester and driver populations for reciprocal SSH procedures. Positive clones from each of the two libraries were randomly selected, and dot blotting was performed for the analysis of expression patterns. A total of 513 clones from the phosphate-replete library and 423 clones from the phosphate-limited library were sequenced, assembled, and compared to sequences in GenBank using BLASTX. Of the 103 differentially expressed gene fragments from the phosphate-replete library, 34% showed significant homology to other known proteins, while only 23% of the 65 differentially expressed gene fragments from the phosphate-limited library showed homology to other proteins. To further assess mRNA expression, real-time RT-PCR analysis was employed and expression profiles were generated over a 14-day time course for three clones from the phosphate-replete library and five clones from the phosphate-limited library. The fragments isolated provide the basis for future cloning of full-length genes and functional analysis.     

Energetic basis for selective recognition of T*G mismatched base pairs in DNA by imidazole-rich polyamides

To complement available structure and binding results and to develop a detailed understanding of the basis for selective molecular recognition of T·G mismatches in DNA by imidazole containing polyamides, a full thermodynamic profile for formation of the T·G–polyamide complex has been determined. The amide-linked heterocycles f-ImImIm and f-PyImIm (where f is formamido group, Im is imidazole and Py is pyrrole) were studied by using biosensor-surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) with a T·G mismatch containing DNA hairpin duplex and a similar DNA with only Watson–Crick base pairs. Large negative binding enthalpies for all of the polyamide–DNA complexes indicate that the interactions are enthalpically driven. SPR results show slower complex formation and stronger binding of f-ImImIm to the T·G than to the match site. The thermodynamic analysis indicates that the enhanced binding to the T·G site is the result of better entropic contributions. Negative heat capacity changes for the complex are correlated with calculated solvent accessible surface area changes and indicate hydrophobic contributions to complex formation. DNase I footprinting analysis in a long DNA sequence provided supporting evidence that f-ImImIm binds selectively to T·G mismatch sites.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Regulation of UvrD Helicase Activity by MutL

Escherichia coli UvrD is a superfamily 1 helicase/translocase involved in multiple DNA metabolic processes including methyl-directed mismatch DNA repair. Although a UvrD monomer can translocate along single-stranded DNA, a UvrD dimer is needed for processive helicase activity in vitro. E. coli MutL, a regulatory protein involved in methyl-directed mismatch repair, stimulates UvrD helicase activity; however, the mechanism is not well understood. Using single-molecule fluorescence and ensemble approaches, we find that a single MutL dimer can activate latent UvrD monomer helicase activity. However, we also find that MutL stimulates UvrD dimer helicase activity. We further find that MutL enhances the DNA-unwinding processivity of UvrD. Hence, MutL acts as a processivity factor by binding to and presumably moving along with UvrD to facilitate DNA unwinding.     

Increase of green plant regeneration efficiency by callus selection in Puccinellia limosa (Schur.) Holmbg.

Three main types of callus have been selected from seeds of salt marsh grass(Puccinellia limosa (Schur.) Holmbg.) subcultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and kinetin. Callus type I differentiated only occasionally. Callus type II produced roots but no shoots under all tested culture conditions. Both green (47 %) and albino plants have been obtained from the embryogenic callus type III. Callus type III was divided into two subtypes (greening and non-greening) according to the presence or absence of green spots. Separated greening embryogenic callus gave up to 87 % green plants, whereas non-greening callus produced only 4 %.Abbreviations ABA Abscisic acid - BA Benzyladenine - CH Casein hydrolysate - CM Coconut milk - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - KIN Kinetin - MS Murashige and Skoog medium - NAA 1-Naphthaleneacetic acid