Regeneration patterns in a Central European dry heathland: effects of burning,sod-cutting and cutting

Vegetation development in dry heathlands was studied during a six-year period following experimental burning, sod-cutting and cutting in a continental area in the southern part of the Czech Republic (Podyjí National Park). Calluna vulgaris-dominated heathlands in the area were composed of uneven-aged stands and had been unmanaged for several decades. The aim of the study was testing the applicability of Western European management systems to nature conservation in a continental area. Species composition was recorded in 8 permanent plots using 25×25 cm grids, and vegetation recovery following different treatments was analysed. Calluna recovered successfully after burning. Burning dense heathlands, which contained a large amount of woody fuel, resulted in a medium-intensity fire that destroyed moss mats and litter and exposed patches of bare ground to facilitate Calluna regeneration by seed, in addition to vegetative regrowth. On the contrary, in open heathlands with patches of herbaceous vegetation, low-intensity fires which failed to expose mineral soil were more typical. Almost all Calluna regeneration was vegetative in this case, and regrowth was slower. Heathland recovery after sod-cutting to mineral soil depended on whether or not Calluna seed germination occurred in the plot. With germination, the community developed towards heathland; without towards grassland. Cutting promoted a striking increase in grass cover, which was followed by the slow recovery of Calluna. The experiments suggest that of these management systems, burning is perhaps the most appropriate in the study area. A nature conservation management system is proposed, based on sheep grazing combined with rotational burning of restricted patches.     

Monoclonal antibody against a peptide of human prion protein discriminates between Creutzfeldt-Jacob's disease-affected and normal brain tissue

Current methods for diagnosing transmissible spongiform encephalopathies rely on the degradation of the cellular prion protein (PrP(C)) and the subsequent detection of the protease-resistant remnant of the pathological prion isoform PrP(Sc) by antibodies that react with all forms of PrP. We report on a monoclonal antibody, V5B2, raised against a peptide from the C-terminal part of PrP, which recognizes an epitope specific to PrP(Sc). In cryostat sections from Creutzfeldt-Jacob's disease (CJD) patients' brains, V5B2 selectively labels various deposits of PrP(Sc) without any pretreatment for removal of PrP(C). V5B2 does not bind to non-CJD brain samples or to recombinant PrP, either in its native or denatured form. Specificity for PrP is confirmed by a sandwich enzyme-linked immunosorbent assay utilizing V5B2, which discriminates between CJD and normal samples without proteinase K treatment, and by immunoprecipitation from CJD brain homogenate. The PrP(Sc)-specific epitope is disrupted by denaturation. We conclude that the C-terminal part of PrP in disease-associated PrP(Sc) aggregates forms a structural epitope whose conformation is distinct from that of PrP(C).     

The alpha 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart

The primary objective of this study was to examine the functional role of the Na,K-ATPase alpha 1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the alpha 1 or alpha 2 isoform gene exhibit opposite phenotypes. Hearts from alpha 2(+/-) animals are hypercontractile, whereas those of the alpha 1(+/-) animals are hypocontractile. The cardiac phenotype of the alpha 1(+/-) animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive alpha 1 isoform and a ouabain-resistant alpha 2 isoform of the Na,K-ATPase, and we analyzed cardiac contractility following selective inhibition of the alpha1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the alpha 1 isoform without affecting the activity of the alpha 2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified alpha 1 and alpha 2 isoforms. Pretreatment with 10 microm KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the alpha1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The alpha 1 isoform co-immunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the alpha 1 isoform regulates cardiac contractility, and that both the alpha 1 and alpha 2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart.     

Experimental tests of the cellular tensegrity hypothesis

The tensegrity model depicts the cytoskeleton (CSK) as a prestressed network of interconnected filaments. The prestress is generated by the CSK contractile apparatus and is partly balanced by traction at the cell-substrate interface and partly by CSK internal compression elements such as microtubules (MTs). A key feature of tensegrity is that the shear modulus (G) must increase in proportion with the prestress. Here we have tested that prediction as well as the idea that compression of MTs balance a portion of the cell prestress. Airway smooth muscle cells were studied. Traction microscopy was used to calculate traction. Because traction must be balanced by the stress within the cell, the prestress could be computed. Cell G was measured by oscillatory magnetic cytometry. The prestress was modulated using graded concentrations of contracting (histamine) or relaxing (isoproterenol) agonists and by disrupting MTs by colchicine. It was found that G increased in proportion with the prestress and that compression of MTs balanced a significant, but a relatively small fraction of the prestress. Taken together, these results do not disprove other models of cell deformability, nor they prove tensegrity. However, they do support a priori predictions of tensegrity. As such, it may not be necessary to invoke more complex mechanisms to explain these central features of cell deformability.     

Effects of war aggression in Croatia on histopathological manifestations of breast cancer in defined population of one county

The aim of the present study was to determine the differences in epidemiological and clinical manifestations of breast cancer during the war in Croatia and in peacetime. 660 consecutive patients were recorded (656 female and 4 male patients) from Pozesko-Slavonska County. The changes in histopathological features were recorded in war period (1991-1995, 156 patients) and through two control periods, before the war (1981-1990, 282 patients) and after the war (1995-2000, first five months, 223 patients). The relative predictive value was calculated using chi 2-test. The survival was calculated according to Kaplan-Meier analysis of survival. The histopathological analysis showed an equal distribution of noninvasive cancer (in situ cancer) across periods. In the war period, the level of the most common invasive cancer, ductal breast cancer, was lower (57.7%), compared to control periods (71.2%:63.7%:68.2%). Opposite to that, invasive lobular cancer was more common in the war period (3.2%), compared to control periods (0.7%-1.3%). Furthermore, mixed cancer was also increased in the war period (7.1%) compared to control periods (0.7%-2.2%), as was medullar cancer (10.9% vs. 5.5%-5.9%). The study showed statistically significant differences in the survival of patients with different histopathological diagnoses (Log Rank = 47.49, df = 7, p < 0.0001), while the histological grade of tumor, as a predictive factor was not proved to be statistically significant (p > 0.05). This study confirmed the influence of war of war on histopathological incidence of some forms of breast cancer.     

Molecular characterisation of Neoparamoeba strains isolated from gills of Scophthalmus maximus

Small subunit ribosomal RNA gene sequences were determined for 5 amoeba strains of the genus Neoparamoeba Page, 1987 that were isolated from gills of Scophthalmus maximus (Linnaeus, 1758). Phylogenetic analyses revealed that 2 of 5 morphologically indistinguishable strains clustered with 6 strains identified previously as N. pemaquidensis (Page, 1970). Three strains branched as a clade separated from N. pemaquidenis and N. aestuarina (Page, 1970) clades. Our analyses suggest that these 3 strains could be representatives of an independent species. In a more comprehensive eukaryotic tree, strains belonging to Neoparamoeba spp. formed a monophyletic group with a sister-group relationship to Vannella anglica Page, 1980. They did not cluster with Gymnamoebae of the families Hartmannellidae, Flabellulidae, Leptomyxidae or Amoebidae presently available in GenBank.     

Sample Power and ExpDesign: tools for improving design of animal experiments

Proper experimental design, involving the correct number of animals, should be a basic skill for any scientist working with animals. The authors describe a university-developed and freely available tutorial program and an interactive computer-assisted learning program, both of which guide students through the steps necessary for designing animal experiments and estimating optimal sample sizes.     

Cyclin-dependent kinase inhibitor,roscovitine, in combination with exogenous cytokinin,N6-benzyladenine,causes increase of cis-cytokinins in immobilized tobacco cells

Dynamics of the response of tobacco cells (line BY-2) to exogenous cytokinin, N ⁶-benzyladenine, and cyclin-dependent kinase inhibitor, roscovitine, was followed using alginate-immobilized cells packed into a column. N ⁶-Benzyladenine (1.25 M) increased the synthesis of the physiologically-active endogenous cytokinin, isopentenyladenosine, in the effluent up to 0.1 nM. Simultaneously, conversion of the excess of endogenous cytokinins to biologically inactive derivatives of cis-zeatin occurred, up to 0.8 nM. Roscovitine (50 M) further increased cis-cytokinins, up to 2.2 nM.     

Circular dichroism spectroscopy of conformers of (guanine + adenine) repeat strands of DNA

(Guanine+adenine) strands of DNA are known to associate into guanine tetraplexes, homodimerize into parallel or antiparallel duplexes, and fold into a cooperatively melting single strand resembling the protein alpha helix. Using CD spectroscopy and other methods, we studied how this conformational polymorphism depended on the primary structure of DNA. The study showed that d(GGGA)(5) and d(GGA)(7) associated into homoduplexes at low salt or in the presence of LiCl but were prone to guanine tetraplex formation, especially in the presence of KCl. In addition, they yielded essentially the same CD spectrum in the presence of ethanol as observed with the ordered single strand of d(GA)(10). Strands of d(GA)(10), d(GGAA)(5), d(GAA)(7), and d(GAAA)(5) associated into homoduplexes in both LiCl and KCl solutions, but not into guanine tetraplexes. d(GAAA)(5) and d(GAA)(7) further failed to form the single-stranded conformer in aqueous ethanol. Adenine protonation, however, stabilized the single-stranded conformer even in these adenine-rich fragments. The ordered single strands, homoduplexes as well as the guanine tetraplexes, all provided strikingly similar CD spectra, indicating that all of the conformers shared similar base stacking geometries. The increasing adenine content only decreased the conformer thermostability.     

Extracellular ribonuclease from Bacillus pumilus]

The extracellular ribonuclease (RNAse Bp) was isolated from the cultural medium filtrate of Bacillus pumilus by ammonium sulfate precipitation and two stages of ion-exchange chromatography on carboxymethyl- and phospho-cellulose columns. The amino acid composition and N-terminal amino acid residue have been determined. The kinetic parameters of cleavage reaction of synthetic polynucleotides have been measured. According to their structural homology RNAse Bp has been shown to be similar to RNAses Ba and Bi. Catalytic properties of the enzyme are very close to RNAse Bi.