Th1 and Th2 cytokine profile in patients with early onset periodontitis and their healthy siblings

Early onset periodontitis (EOP) is a chronic inflammatory periodontal disease with a strong genetic link affecting individuals aged 17 to 25. In the familial studies we tested the hypothesis about the role of Th1 and Th2 cytokines in the pathogenesis of EOP disease. The study involved 6 individuals with EOP disease and their 6 siblings with healthy periodontium. Actinobacillus actinomycetemcomitans (A. a), a bacterium typical for EOP, was detected in all people studied. Th1 and Th2 cytokine production was measured after in vitro stimulation. Peripheral blood mononuclear cells (PBMC) were isolated and cultivated for 24 h and 7 days with PWM, A. a. or Escherichia coli. The levels of IL-4, IFN-gamma, IgA, IgG and IgM were measured by ELISA methods. After in vitro stimulation of PBMC, a significantly higher production of IL-4 and significantly lower production of IFN-gamma were found in the group of patients compared with their healthy siblings. The increased level of IL-4 in patients was in good agreement with an increased level of IgM after stimulation of lymphocytes with E. coli. These results support Seymour's hypothesis according to which patients with progressive disease primarily activate Th2 lymphocytes while non-susceptible individuals activate Th1 lymphocytes.     

Phytoremediation: novel approaches to cleaning up polluted soils

Environmental pollution with metals and xenobiotics is a global problem, and the development of phytoremediation technologies for the plant-based clean-up of contaminated soils is therefore of significant interest. Phytoremediation technologies are currently available for only a small subset of pollution problems, such as arsenic. Arsenic removal employs naturally selected hyperaccumulator ferns, which accumulate very high concentrations of arsenic specifically in above-ground tissues. Elegant two-gene transgenic approaches have been designed for the development of mercury or arsenic phytoremediation technologies. In a plant that naturally hyperaccumulates zinc in leaves, approximately ten key metal homeostasis genes are expressed at very high levels. This outlines the extent of change in gene activities needed in the engineering of transgenic plants for soil clean-up. Further analysis and discovery of genes for phytoremediation will benefit from the recent development of segregating populations for a genetic analysis of naturally selected metal hyperaccumulation in plants, and from comprehensive ionomics data--multi-element concentration profiles from a large number of Arabidopsis mutants.     

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.     

Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation

As a response to hyperosmotic stress bacterial cells accumulate compatible solutes by synthesis or by uptake. Beside the instant activation of uptake systems after an osmotic upshift, transport systems show also a second, equally important type of regulation. In order to adapt the pool size of compatible solutes in the cytoplasm to the actual extent of osmotic stress, cells down-regulate solute uptake when the initial osmotic stress is compensated. Here we describe the role of the betaine transporter BetP, the major uptake carrier for compatible solutes in Corynebacterium glutamicum, in this adaptation process. For this purpose, betP was expressed in cells (C. glutamicum and Escherichia coli), which lack all known uptake systems for compatible solutes. Betaine uptake mediated by BetP as well as by a truncated form of BetP, which is deregulated in its response to hyperosmotic stress, was dissected into the individual substrate fluxes of unidirectional uptake, unidirectional efflux and net uptake. We determined a strong decrease of unidirectional betaine uptake by BetP in the adaptation phase. The observed decrease in net uptake was thus mainly due to a decrease of Vmax of BetP and not a consequence of the presence of separate efflux system(s). These results indicate that adaptation of BetP to osmotic compensation is different from activation by osmotic stress and also different from previously described adaptation mechanisms in other organisms. Cytoplasmic K+, which was shown to be responsible for activation of BetP upon osmotic stress, as well as a number of other factors was ruled out as triggers for the adaptation process. Our results thus indicate the presence of a second type of signal input in the adaptive regulation of osmoregulated carrier proteins.     

Biased exon/intron distribution of cryptic and de novo 3' splice sites

We compiled sequences of previously published aberrant 3′ splice sites (3′ss) that were generated by mutations in human disease genes. Cryptic 3′ss, defined here as those resulting from a mutation of the 3′YAG consensus, were more frequent in exons than in introns. They clustered in ~20 nt region adjacent to authentic 3′ss, suggesting that their under-representation in introns is due to a depletion of AG dinucleotides in the polypyrimidine tract (PPT). In contrast, most aberrant 3′ss that were induced by mutations outside the 3′YAG consensus (designated ‘de novo’) were in introns. The activation of intronic de novo 3′ss was largely due to AG-creating mutations in the PPT. In contrast, exonic de novo 3′ss were more often induced by mutations improving the PPT, branchpoint sequence (BPS) or distant auxiliary signals, rather than by direct AG creation. The Shapiro–Senapathy matrix scores had a good prognostic value for cryptic, but not de novo 3′ss. Finally, AG-creating mutations in the PPT that produced aberrant 3′ss upstream of the predicted BPS in vivo shared a similar ‘BPS-new AG’ distance. Reduction of this distance and/or the strength of the new AG PPT in splicing reporter pre-mRNAs improved utilization of authentic 3′ss, suggesting that AG-creating mutations that are located closer to the BPS and are preceded by weaker PPT may result in less severe splicing defects.     

Wildflower areas within revitalized agricultural matrices boost small mammal populations but not breeding Barn Owls

Agro-ecosystems have recently experienced dramatic losses of biodiversity due to more intensive production methods. In order to increase species diversity, agri-environment schemes provide subsidies to farmers who devote a fraction of their land to ecological compensation areas (ECAs). Several studies have shown that invertebrate biodiversity is actually higher in ECAs than in nearby intensively cultivated farmland. It remains poorly understood, however, to what extent ECAs also favour vertebrates, such as small mammals and their predators, which would contribute to restoring functional food chains within revitalised agricultural matrices. We studied small mammal populations among eight habitat types—including wildflower areas, a specific ECA in Switzerland—and habitat selection (radiotracking) by the Barn Owl Tyto alba, one of their principal predators. Our prediction was that habitats with higher abundances of small mammals would be more visited by foraging Barn Owls during the period of chicks’ provisioning. Small mammal abundance tended to be higher in wildflower areas than in any other habitat type. Barn Owls, however, preferred to forage in cereal fields and grassland. They avoided all types of crops other than cereals, as well as wildflower areas, which suggests that they do not select their hunting habitat primarily with respect to prey density. Instead of prey abundance, prey accessibility may play a more crucial role: wildflower areas have a dense vegetation cover, which may impede access to prey for foraging owls. The exploitation of wildflower areas by the owls might be enhanced by creating open foraging corridors within or around wildflower areas. Wildflower areas managed in that way might contribute to restore functional links in food webs within agro-ecosystems.     

Activation and modulation of ligand-gated ion channels

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.     

Cold-active enzymes studied by comparative molecular dynamics simulation

Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included α-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein. Figure Collective motions in C^α atoms of the active site of cold-active xylanase Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.