Fibronectin binding and cell-surface hydrophobicity of attaching effacing enteropathogenic Escherichia coli strains isolated from newborn and weanling rabbits with diarrhoea

Abstract 32 different strains of Escherichia coli isolated from rabbits with diarrhoea were studied for cell-surface properties which may be involved in intestinal colonisation. Strains isolated from diarrhoeic suckling (6 strains) and weaning (26 strains) rabbits which were shown to attach to brush borders in vivo, showed high relative cell-surface hydrophobicity as determined by the Salt Aggregation Test (SAT) when grown on Colonisation Factor Antigen (CFA) agar at 33°C. Cells of these strains grown to express surface hydrophobicity were also defined as high, moderate or low binders of ¹²⁵I-fibronectin or its ¹²⁵I-29-kDa fragment in a standard binding assay. Based on these findings, we propose that binding to intestinal cell surface (mucus)-associated fibronectin may be an early important step in intestinal colonisation of the small bowel in enteropathogenic E. coli (EPEC) diarrhoea in rabbits and other animal species.     

Chromophore distortions in the bacteriorhodopsin photocycle: evolution of the H-C14-C15-H dihedral angle measured by solid-state NMR.

In recent years, structural information about bacteriorhodopsin has grown substantially with the publication of several crystal structures. However, precise measurements of the chromophore conformation in the various photocycle states are still lacking. This information is critical because twists about the chromophore backbone chain can influence the Schiff base nitrogen position, orientation, and proton affinity. Here, we focus on the C14-C15 bond, using solid-state nuclear magnetic resonance spectroscopy to measure the H-C14-C15-H dihedral angle. In the resting state (bR(568)), we obtain an angle of 164 +/- 4 degrees, indicating a 16 degrees distortion from a planar all-trans chromophore. The dihedral angle is found to decrease to 147 +/- 10 degrees in the early M intermediate (M(o)) and to 150 +/- 4 degrees in the late M intermediate (M(n)). These results demonstrate changes in the chromophore conformation undetected by recent X-ray diffraction studies.     

Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis

Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.     

The Net Landscape Carbon Balance—Integrating terrestrial and aquatic carbon fluxes in a managed boreal forest landscape in Sweden

The boreal biome exchanges large amounts of carbon (C) and greenhouse gases (GHGs) with the atmosphere and thus significantly affects the global climate. A managed boreal landscape consists of various sinks and sources of carbon dioxide (CO₂), methane (CH₄), and dissolved organic and inorganic carbon (DOC and DIC) across forests, mires, lakes, and streams. Due to the spatial heterogeneity, large uncertainties exist regarding the net landscape carbon balance (NLCB). In this study, we compiled terrestrial and aquatic fluxes of CO₂, CH₄, DOC, DIC, and harvested C obtained from tall‐tower eddy covariance measurements, stream monitoring, and remote sensing of biomass stocks for an entire boreal catchment (~68 km²) in Sweden to estimate the NLCB across the land–water–atmosphere continuum. Our results showed that this managed boreal forest landscape was a net C sink (NLCB = 39 g C m^?2 year^?1) with the landscape–atmosphere CO₂ exchange being the dominant component, followed by the C export via harvest and streams. Accounting for the global warming potential of CH₄, the landscape was a GHG sink of 237 g CO₂‐eq m^?2 year^?1, thus providing a climate‐cooling effect. The CH₄ flux contribution to the annual GHG budget increased from 0.6% during spring to 3.2% during winter. The aquatic C loss was most significant during spring contributing 8% to the annual NLCB. We further found that abiotic controls (e.g., air temperature and incoming radiation) regulated the temporal variability of the NLCB whereas land cover types (e.g., mire vs. forest) and management practices (e.g., clear‐cutting) determined their spatial variability. Our study advocates the need for integrating terrestrial and aquatic fluxes at the landscape scale based on tall‐tower eddy covariance measurements combined with biomass stock and stream monitoring to develop a holistic understanding of the NLCB of managed boreal forest landscapes and to better evaluate their potential for mitigating climate change.     

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.     

Nicastrin, presenilin, APH-1, and PEN-2 form active gamma-secretase complexes in mitochondria

Mitochondria are central in the regulation of cell death. Apart from providing the cell with ATP, mitochondria also harbor several death factors that are released upon apoptotic stimuli. Alterations in mitochondrial functions, increased oxidative stress, and neurons dying by apoptosis have been detected in Alzheimer's disease patients. These findings suggest that mitochondria may trigger the abnormal onset of neuronal cell death in Alzheimer's disease. We previously reported that presenilin 1 (PS1), which is often mutated in familial forms of Alzheimer's disease, is located in mitochondria and hypothesized that presenilin mutations may sensitize cells to apoptotic stimuli at the mitochondrial level. Presenilin forms an active gamma-secretase complex together with Nicastrin (NCT), APH-1, and PEN-2, which among other substrates cleaves the beta-amyloid precursor protein (beta-APP) generating the amyloid beta-peptide and the beta-APP intracellular domain. Here we have identified dual targeting sequences (for endoplasmic reticulum and mitochondria) in NCT and showed expression of NCT in mitochondria by immunoelectron microscopy. We also showed that NCT together with APH-1, PEN-2, and PS1 form a high molecular weight complex located in mitochondria. gamma-secretase activity in isolated mitochondria was demonstrated using C83 (alpha-secretase-cleaved C-terminal 83-residue beta-APP fragment from BD8 cells lacking presenilin and thus gamma-secretase activity) or recombinant C100-Flag (C-terminal 100-residue beta-APP fragment) as substrates. Both systems generated an APP intracellular domain, and the activity was inhibited by the gamma-secretase inhibitors l-685,458 or Compound E. This novel localization of NCT, PS1, APH-1, and PEN-2 expands the role and importance of gamma-secretase activity to mitochondria.     

Fine mapping and imprinting analysis for fatness trait QTLs in pigs

Quantitative trait loci (QTL) for fatness traits were reported recently in an experimental Meishan × Large White and Landrace F₂ cross. To further investigate the regions on pig Chr 2 (SSC2), SSC4, and SSC7, 25 additional markers from these regions were typed on 800 animals (619 F₂ animals, their F₁ parents, and F₀ grandfathers). Compared with the published maps, a modified order of markers was observed for SSC4 and SSC7. QTL analyses were performed both within the half-sib families as well as across families (line cross). Furthermore, a QTL model accounting for imprinting effects was tested. Information content could be increased considerably on all three chromosomes. Evidence for the backfat thickness QTL on SSC7 was increased, and the location could be reduced to a 33-cM confidence interval. The QTL for intramuscular fat on SSC4 could not be detected in this half-sib analysis, whereas under the line cross model a suggestive QTL on a different position on SSC4 was detected. For SSC2, in the half-sib analysis, a suggestive QTL for backfat thickness was detected with the best position at 26 cM. Imprinting analysis, however, revealed a genome-wise, significant, paternally expressed QTL on SSC2 with the best position at 63 cM. Our results suggest that this QTL is different from the previously reported paternally expressed QTL for muscle mass and fat deposition on the distal tip of SSC2p. Received: 15 October 1999 / Accepted: 21 February 2000     

CD7 is a differentiation marker that identifies multiple CD8 T cell effector subsets

The adaptive immune response of human CD8 T cells to invading pathogens involves the differentiation of naive cells into memory and effector cells. However, the lineage relationship between memory and effector cells and the differentiation of CD8 T cells into distinct subsets of effector cell subpopulations are subjects of considerable debate. CD7 identifies three populations of CD8 T cells: CD7 high (CD7(high)), low (CD7(low)), and negative (CD7(neg)) that translate into subsets with distinct functional properties. The CD7(high) subset contains naive and memory cells and the CD7(low) and CD7(neg) subsets contain effector cells. The effector cells can functionally be divided into cytokine-secreting effector CD8 T cells and lytic effector CD8 T cells. These data provide a model of human CD8 T cell differentiation in which specialized distinct subpopulations can be identified by expression of CD7.     

Prediction of partial membrane protein topologies using a consensus approach

We have developed a method to reliably identify partial membrane protein topologies using the consensus of five topology prediction methods. When evaluated on a test set of experimentally characterized proteins, we find that approximately 90% of the partial consensus topologies are correctly predicted in membrane proteins from prokaryotic as well as eukaryotic organisms. Whole-genome analysis reveals that a reliable partial consensus topology can be predicted for approximately 70% of all membrane proteins in a typical bacterial genome and for approximately 55% of all membrane proteins in a typical eukaryotic genome. The average fraction of sequence length covered by a partial consensus topology is 44% for the prokaryotic proteins and 17% for the eukaryotic proteins in our test set, and similar numbers are found when the algorithm is applied to whole genomes. Reliably predicted partial topologies may simplify experimental determinations of membrane protein topology.     

Influence of ploidy level on morphology,growth and drought susceptibility in <Emphasis Type="Italic">Spathiphyllum wallisii</Emphasis>

In this study, we analysed morphological, anatomical and physiological effects of polyploidisation in Spathiphyllum wallisii in order to evaluate possible interesting advantages of polyploids for ornamental breeding. Stomatal density was negatively correlated with increased ploidy level. Stomatal size increased in polyploids. Tetraploid Spathiphyllum plants had more ovate and thicker leaves. The inflorescence of tetraploids had a more ovate and thicker spathum, a more cylindrical spadix and a thicker but shorter flower stalk. Biomass production of the tetraploids was reduced, as expressed by lower total dry weights, and tetraploids produced fewer shoots and leaves compared with their diploid progenitors. Furthermore, tetraploid Spathiphyllum plants were more resistant to drought stress compared with diploid plants. After 15 days of drought stress, diploids showed symptoms of wilting, while the tetraploids showed almost no symptoms. Further, measurements of stomatal resistance, leaf water potential, relative water content and proline content indicated that the tetraploid genotypes were more resistant to drought stress compared with the diploids.