Comparative genomic hybridisation and ultrafast pyrosequencing revealed remarkable differences between the Sinorhizobium meliloti genomes of the model strain Rm1021 and the field isolate SM11

Genomic variation between the Sinorhizobium meliloti model strain Rm1021 and the field isolate SM11 was assessed by using the genome-wide S. meliloti Rm1021 Sm6k-oligonucleotide microarray in a comparative genomic hybridisation experiment. Several gene clusters present in the Rm1021 genome are missing in the SM11 genome. In detail, three missing gene clusters were identified for the chromosome, five for megaplasmid pSymA and two for megaplasmid pSymB. To confirm these hybridisation results, the draft genome sequence of the S. meliloti field isolate SM11 was established by 454-pyrosequencing. Three sequencing runs on the ultrafast Genome Sequencer 20 System yielded 112.5 million bases. These could be assembled into 905 larger contigs resulting in a nearly 15-fold coverage of the 7.1Mb SM11 genome. The missing gene regions identified by comparative genomic hybridisation could be confirmed by the results of the 454-sequencing project. An in-depth analysis of these gene regions resulted in the following findings: (i) a complete type I restriction/modification system encoded by a composite transposon is absent in the chromosome of strain SM11. (ii) Most of the Rm1021 denitrification genes and the complete siderophore biosynthesis operon were found to be missing on SM11 megaplasmid pSymA. (iii) S. meliloti SM11 megaplasmid pSymB lacks a complete cell surface carbohydrate synthesis gene cluster. (iv) Several genes that are absent in the SM11 genome could be assigned to insertion sequences and transposons.     

Comparing soluble and co-immobilized catalysts for 2-ketoaldose production by pyranose 2-oxidase and auxiliary enzymes

The tri-enzyme system pyranose 2-oxidase (P2O), laccase, and catalase was used to study major parameters in the homogeneous and heterogeneous application of a multi-component enzymatic machinery. P2O oxidizes aldoses to 2-ketosugars, which are interesting intermediates in carbohydrate chemistry, and concomitantly reduces oxygen or alternative electron acceptors. The enzyme was immobilized on eleven agarose or acrylic resins using various coupling methods. The binding capacity was determined and an acrylic carrier with the most suitable properties selected for detailed studies. As P2O shows higher turnover numbers with the electron acceptor 1,4-benzoquinone than with oxygen, the use of this alternative electron acceptor was enabled by employing laccase for the continuous reoxidation of hydroquinone. The laccase regeneration system was found to increase the specific productivity up to 3-fold. Catalase was used to disproportionate the formed hydrogen peroxide in close proximity to the oxygen consuming enzymes and applied in different amounts to adjust the hydrogen peroxide concentration, which was found to be the main reason for enzyme deactivation under turnover conditions. In contrast to homogeneous catalysis, the specific productivity of heterogeneous catalysts under the applied experimental conditions was limited primarily by oxygen transfer, an effect significantly reduced by the laccase regeneration system.     

Protein p0071, a major plaque protein of non-desmosomal adhering junctions,is a selective cell-type marker

Protein p0071, which originally was introduced as a member of the p120-subfamily of armadillo proteins, common to desmosomes and adhaerens junctions (AJs) and to several other cell structures (centrosomes, midbodies), has been localized by using a series of novel mono- and polyclonal antibodies generated against various domains of the molecule. By protein analysis and immunolocalization techniques, protein p0071 has been localized as a plaque protein in AJs of diverse epithelia and certain vascular endothelia, in the composite junctions (areal compositae) of the intercalated disks of cardiomyocytes, and in the punctate or more extended AJs of the vast majority of cell culture types examined, including mitotic states. Using these antibodies, we have also shown that this AJ protein occurs only rarely or is even absent in tissues such as skeletal and smooth muscles, in a series of mesenchymal tissue cells, and in specific desmosome-rich cells such as those of the upper layers of the epidermis and certain other stratified epithelia and Hassall corpuscles of the thymus. We have also demonstrated that p0071 is absent from desmosomes. The occurrence of two major subtypes of lymphatic endothelial cells, one with AJs containing p0071 and one without detectable p0071, is emphasized. Possible structural and functional roles of p0071 are discussed in light of these new findings regarding its localization, and the addition of p0071 to the armamentarium of cytodiagnostic cell-type markers is recommended. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by project grants from the German Ministry for Education and Research (BMBF) in a cooperative research program entitled “Standardization of mesenchymal stem cells for regenerative medicine (START-MSC)” and from the Deutsche Krebshilfe (project 10–2049) to W.W. Franke.     

Subtypes of melanocytes and melanoma cells distinguished by their intercellular contacts: heterotypic adherens junctions,adhesive associations,and dispersed desmoglein 2 glycoproteins

In the tissue integration of melanocytes and melanoma cells, an important role is attributed to cell adhesion molecules, notably the cadherins. In cultured melanoma cells, we have previously described a more heterogeneous repertoire of cadherins than normal, including some melanoma subtypes synthesizing the desmosomal cadherin, desmoglein 2, out of the desmosomal context. Using biochemical and immunological characterization of junctional molecules, confocal laser scanning, and electron and immunoelectron microscopy, we now demonstrate homo- and heterotypic cell-cell adhesions of normal epidermal melanocytes. In human epidermis, both in situ and in cell culture, melanocytes and keratinocytes are connected by closely aligned membranes that are interspersed by small puncta adhaerentia containing heterotypic complexes of E- and P-cadherin. Moreover, melanocytes growing in culture often begin to synthesize desmoglein 2, which is dispersed over extended areas of intimate adhesive cell-cell associations. As desmoglein 2 is not found in melanocytes in situ, we hypothesize that its synthesis is correlated with cell proliferation. Indeed, in tissue microarrays, desmoglein 2 has been demonstrated in a sizable subset of nevi and primary melanomas. The biological meanings of these cell-cell adhesion molecule arrangements, the possible diagnostic and prognostic significance of these findings, and the implications of the heterogeneity types of melanomas are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported in parts by grants from the Deutsche Forschungsgemeinschaft to W. K. Peitsch (project PE 896/1) and the Deutsche Krebshilfe to W. W. Franke (project 10-2049).     

Fluoreszenz-in-situ-Hybridisierung in der humangenetischen Diagnostik

The field of genetic diagnostics incorporates a variety of methods that complement each other. Therefore, the development of new methods calls for a review of the advantages and limitations of established and new technologies. Fluorescence in situ hybridization (FISH) is routinely applied in genetics. Custom-designed and commercially available probes allow for nearly unlimited and targeted visualization of genomic DNA using either metaphase spreads, interphase nuclei, tissue sections, or living cells. FISH applications are particularly important for the detection of structural rearrangements such as microdeletions, translocations, inversions, and insertions, as well as for identification of marker chromosomes, characterization of chromosome breakpoints, and prenatal aneuploidy testing. Furthermore, the analysis of genetic heterogeneity, including mosaicism, is accomplished by evaluating single cells. FISH may also be combined with fluorescent antibodies against cell surface markers and correlated to specific morphologic features of cells and tissues.     

Long-term changes in tree-ring–climate relationships at Mt. Patscherkofel (Tyrol,Austria) since the mid-1980s

Although growth limitation of trees at Alpine and high-latitude timberlines by prevailing summer temperature is well established, the loss of thermal response of radial tree growth during last decades has repeatedly been addressed. We examined long-term variability of climate–growth relationships in ring width chronologies of Stone pine (Pinus cembra L.) by means of moving response functions (MRF). The study area is situated in the timberline ecotone (ca. 2,000–2,200 m a.s.l.) on Mt. Patscherkofel (Tyrol, Austria). Five site chronologies were developed within the ecotone with constant sample depth (≥19 trees) throughout most of the time period analysed. MRF calculated for the period 1866–1999 and 1901–1999 for ca. 200- and ca. 100-year-old stands, respectively, revealed that mean July temperature is the major and long-term stable driving force of Pinus cembra radial growth within the timberline ecotone. However, since the mid-1980s, radial growth in timberline and tree line chronologies strikingly diverges from the July temperature trend. This is probably a result of extreme climate events (e.g. low winter precipitation, late frost) and/or increasing drought stress on cambial activity. The latter assumption is supported by a <10% increase in annual increments of ca. 50-year-old trees at the timberline and at the tree line in 2003 compared with 2002, when extraordinary hot and dry conditions prevailed during summer. Furthermore, especially during the second half of the twentieth century, influence of climate variables on radial growth show abrupt fluctuations, which might also be a consequence of climate warming on tree physiology.     

Susceptibility of four inbred mouse strains to a low-pathogenic isolate of <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>

EUMORPHIA (European Union Mouse Research for Public Health and Industrial Application) is a research program involved in developing new approaches in phenotyping, mutagenesis, and informatics to improve characterization of mouse models for understanding human physiology and disease. Secondary screen experiments include the development of assays to identify mice with altered susceptibility or resistance to infections. In this context we developed a new model and established a standard operating procedure for the experimental infection of mice with Yersinia (Y.) enterocolitica. In contrast with previous studies that dealt with high-pathogenic Y. enterocolitica, we used the low-pathogenic Y. enterocolitica strain E40 to analyze differences in the immune response of four strains of inbred mice (BALB/c, C3H/HeN, 129P2, C57BL/6) after oral infection. The determination of colony-forming units in Peyer’s patches and histologic analysis supported the observations that BALB/c are less able to ameliorate the infection within 21 days. The immune defense of C57BL/6 mice against Yersinia was the most effective resulting in a nearly complete elimination of bacteria after 21 days. C3H/HeN and 129P2 were intermediate. Analysis of serum immunoglobulins (Ig) by Luminex showed a significant increase of IgG2b levels 21 days after infection in all four inbred strains. The other immunoglobulins remained nearly constant. Our infection model discriminates between the efficiency of an infection at an early time point (3 days) and immunity at a later time point (21 days). It is furthermore an appropriate model to characterize genetic differences in resistance and immunity of inbred and mutant mouse lines.     

Ceramic-based multisite microelectrode arrays for simultaneous measures of choline and acetylcholine in CNS

A ceramic-based microelectrode array (MEA) with enzyme coatings for the accurate measurement of acetylcholine (ACh) in brain tissues is presented. Novel design features allow for self-referencing recordings for improved limits of detection and highly selective measurements of ACh and choline (Ch), simultaneously. Design and fabrication features also result in minimal tissue damage during implantation and improved enzyme coatings due to isolated recording sites. In these studies we have used a recombinant human acetylcholinesterase enzyme coating, which has better reproducibility than other commercially available enzymes. The precisely patterned recording site dimensions, low limit of detection (0.2 micro M) and fast response time ( approximately 1s) allow for second-by-second measurements of ACh and Ch in brain tissues. An electropolymerized meta-phenylenediamine (mPD) layer was used to exclude interfering substances from being recorded at the platinum recording sites. Our studies support that the mPD layer was stable for over 24h under in vitro and in vivo recording conditions. In addition, our work supports that the current configuration of the MEAs produces a robust design, which is suited for measures of ACh and Ch in rat brain.     

Frequency-dependent shear impedance of the tectorial membrane

Microscale mechanical probes were designed and bulk-fabricated for applying shearing forces to biological tissues. These probes were used to measure shear impedance of the tectorial membrane (TM) in two dimensions. Forces were applied in the radial and longitudinal directions at frequencies ranging from 0.01-9 kHz and amplitudes from 0.02-4 μN. The force applied was determined by measuring the deflection of the probes’ cantilever arms. TM impedance in the radial direction had a magnitude of 63 ± 28 mN · s/m at 10 Hz and fell with frequency by 16 ± 0.4 dB/decade, with a constant phase of −72 ± 6°. In the longitudinal direction, impedance was 36 ± 9 mN · s/m at 10 Hz and fell by 19 ± 0.4 dB/decade, with a constant phase of −78 ± 4°. Impedance was nearly constant as a function of force except at the highest forces, for which it fell slightly. These results show that the viscoelastic properties of the TM extend over a significant range of audio frequencies, consistent with a poroelastic interpretation of TM mechanics. The shear modulus G′ determined from these measurements was 17-50 kPa, which is larger than in species with a lower auditory frequency range. This value suggests that hair bundles cannot globally shear the TM, but most likely cause bulk TM motion.     

Mitigation of NADH: ubiquinone oxidoreductase deficiency by chronic Trolox treatment

Deficiency of mitochondrial NADH:ubiquinone oxidoreductase (complex I), is associated with a variety of clinical phenotypes such as Leigh syndrome, encephalomyopathy and cardiomyopathy. Circumstantial evidence suggests that increased reactive oxygen species (ROS) levels contribute to the pathogenesis of these disorders. Here we assessed the effect of the water-soluble vitamin E derivative Trolox on ROS levels, and the amount and activity of complex I in fibroblasts of six children with isolated complex I deficiency caused by a mutation in the NDUFS1, NDUFS2, NDUFS7, NDUFS8 or NDUFV1 gene. Patient cells displayed increased ROS levels and a variable decrease in complex I activity and amount. For control cells, the ratio between activity and amount was 1 whereas for the patients this ratio was below 1, indicating a defect in intrinsic catalytic activity of complex I in the latter cells. Trolox treatment dramatically reduced ROS levels in both control and patient cells, which was paralleled by a substantial increase in the amount of complex I. Although the ratio between the increase in activity and amount of complex I was exactly proportional in control cells it varied between 0.1 and 0.8 for the patients. Our findings suggest that the expression of complex I is regulated by ROS. Furthermore, they provide evidence that both the amount and intrinsic activity of complex I are decreased in inherited complex I deficiency. The finding that Trolox treatment increased the amount of complex I might aid the future development of antioxidant treatment strategies for patients. However, such treatment may only be beneficial to patients with a relatively small reduction in intrinsic catalytic defect of the complex.