Detection of Mycobacterium avium subsp. paratuberculosis in two brown bears in the central European Carpathians

The incidence of mycobacterial infections was monitored in brown bears (Ursus arctos) in the National Park Low Tatras in the central European Carpathians in Slovakia. Tissue samples of 20 brown bears were examined microscopically and by culture for the presence of mycobacteria. Acid-fast rods were detected by Ziehl-Neelsen staining in a smear from the kidney of one brown bear, although the culture was negative for mycobacteria. Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis in ruminants, was isolated from the intestinal mucosa of another two brown bears. The isolates were identified by polymerase chain reaction for the specific insertion sequence IS900. Using standardized IS900 restriction fragment length polymorphism (RFLP) analysis, the M. a. paratuberculosis isolates were classified as RFLP type B-C1, which also were detected in the infected cattle in surrounding area. This study describes the first isolation of M. a. paratuberculosis from a brown bear. Our results confirm that animal species other than ruminants can become infected with M. a. paratuberculosis and can act as potential vectors and/or reservoirs of the infection.     

The STAR project: context, objectives and approaches

STAR is a European Commission Framework V project (EVK1-CT-2001-00089). The project aim is to provide practical advice and solutions with regard to many of the issues associated with the Water Framework Directive. This paper provides a context for the STAR research programme through a review of the requirements of the directive and the Common Implementation Strategy responsible for guiding its implementation. The scientific and strategic objectives of STAR are set out in the form of a series of research questions and the reader is referred to the papers in this volume that address those objectives, which include: (a) Which methods or biological quality elements are best able to indicate certain stressors? (b) Which method can be used on which scale? (c) Which method is suited for early and late warnings? (d) How are different assessment methods affected by errors and uncertainty? (e) How can data from different assessment methods be intercalibrated? (f) How can the cost-effectiveness of field and laboratory protocols be optimised? (g) How can boundaries of the five classes of Ecological Status be best set? (h) What contribution can STAR make to the development of European standards? The methodological approaches adopted to meet these objectives are described. These include the selection of the 22 stream-types and 263 sites sampled in 11 countries, the sampling protocols used to sample and survey phytobenthos, macrophytes, macroinvertebrates, fish and hydromorphology, the quality control and uncertainty analyses that were applied, including training, replicate sampling and audit of performance, the development of bespoke software and the project outputs. This paper provides the detailed background information to be referred to in conjunction with most of the other papers in this volume. These papers are divided into seven sections: (1) typology, (2) organism groups, (3) macrophytes and diatoms, (4) hydromorphology, (5) tools for assessing European streams with macroinvertebrates, (6) intercalibration and comparison and (7) errors and uncertainty. The principal findings of the papers in each section and their relevance to the Water Framework Directive are synthesised in short summary papers at the beginning of each section. Additional outputs, including all sampling and laboratory protocols and project deliverables, together with a range of freely downloadable software are available from the project website at www.eu_star.at.     

MDOC and atorvastatin have potential antiinflammatory effects in vascular endothelium of apoE-/- mouse model of atherosclerosis

Members of the immunoglobulin superfamily of endothelial adhesion molecules, vascular cell adhesion molecule (VCAM-1) and intercellular cell adhesion molecule (ICAM- 1), strongly participate in leukocyte adhesion to the endothelium and play an important role in all stages of atherogenesis. The aim of this study was to detect and quantify the changes of endothelial expression of VCAM-1, and ICAM-1 in the vessel wall after the short-term administration of simvastatin, atorvastatin, and micro dispersed derivatives of oxidised cellulose (MDOC™) in apolipoprotein-E-deficient (apoE^−/−) mice atherosclerotic model. Hyperlipidemic apoE^−/− mice (n = 32) received normal chow diet or diet containing simvastatin or atorvastatin 10 mg/kg/day or MDOC™ 50 mg/kg/day. Total cholesterol, VLDL, LDL, HDL and TAG were measured and the endothelial expression of VCAM-1 and ICAM-1 was visualized and quantified by means of immunohistochemistry and stereology, respectively. Total cholesterol levels was insignificantly lowered only in MDOC™ treated mice but not in mice treated with statins. ICAM-1 endothelial expression was not affected by neither simvastatin nor MDOC™ treatment. However, significant diminution of VCAM-1 endothelial expression was observed in both atorvastatin and MDOC™ treated mice. These results provide new information of potential hypolipidemic substance MDOC™ and its potential anti-inflammatory effects. Furthermore, we have confirmed anti-inflammatory effects of atorvastatin independent of plasma cholesterol lowering. Thus, the results of this study show potential benefit of both MDOC™ and atorvastatin treatment in apoE^−/− mouse model of atherosclerosis suggesting their possible combination might be of interest.     

Role of mitochondria in the immune response to cancer: a central role for Ca2+

This study demonstrates that Ca²⁺ stimulates mitochondrial energy metabolism during spleen lymphocyte activation in response to the ascitic Walker 256 tumor in rats. Intracellular Ca²⁺ concentrations, phosphorylated protein kinase C (pPKC) levels, Bcl-2 protein contents, interleukin-2 (IL-2) levels, mitochondrial uncoupling protein-2 (UCP-2) contents and reactive oxygen species (ROS) were significantly elevated in these activated lymphocytes. Mitochondria of activated lymphocytes exhibited high free Ca²⁺ concentrations in the matrix and enhanced oligomycin-sensitive oxygen consumption, indicating an increased rate of oxidative phosphorylation. The production of ROS was largely decreased by diphenylene iodinium in the activated lymphocytes, suggesting that NADPH oxidase is the prevalent source of these species. Accumulation of UCP-2 and the anti-apoptotic protein Bcl-2 is probably important to prevent mitochondrial dysfunction and cell death elicited by the sustained high levels of intracellular Ca²⁺ and ROS and may explain the observed higher resistance from activated lymphocytes against the opening of the mitochondrial membrane permeability pore (MPT). All these changes were blocked by pretreatment of the rats with verapamil, an L-type Ca²⁺ channel antagonist. These data demonstrate a central role of Ca²⁺ in the control of mitochondrial bioenergetics in spleen lymphocytes during the immune response to cancer.     

Proteomic analysis of iron overload in human hepatoma cells

Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.     

The effect of substituted amides of pyrazine-2-carboxylic acids on flavonolignan production in <Emphasis Type="Italic">Silybum marianum</Emphasis> culture <Emphasis Type="Italic">in vitro</Emphasis>

In vitro plant tissue and cell cultures were used to study herbicide effects on growth, selected metabolic activities and other phenomena. The effect of abiotic elicitors, two newly synthesized substituted amides of pyrazine-2-carboxylic acids (synthesized at the Department of Pharmaceutical Chemistry and Drug Control, School of Pharmacy in Hradec Kralove), on the flavonolignan accumulation in callus and suspension culture of Silybum marianum (L.) Gaertn. was investigated. The compounds markedly influenced production of flavonolignans in an in vitro culture. Particularly after the elicitation with a solution of compound 3-methylamide 5-tert-butylpyrazine-2-carboxylic acid at a concentration of 3.71×10⁻⁷ mol 1⁻¹ and within 72 h of elicitation, an increase in flavonolignan production by 893 % in suspension culture versus control took place. The flavonolignan accumulation in callus culture after the elicitation with a solution of 5-brom-2-hydroxyphenylamide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid was also increased by about 1039% (24 hour elicitation and concentration of 2.59×10⁻⁴ mol 1⁻¹).     

Intrinsically Disordered Enamel Matrix Protein Ameloblastin Forms Ribbon-like Supramolecular Structures via an N-terminal Segment Encoded by Exon 5

Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.