Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen Matrices

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.     

Leaf gas exchange of trees in old-growth and young secondary forest stands in Sulawesi, Indonesia

In the tropics, old-growth forests are converted to other land cover types at a high rate and young secondary forest may gain in importance. Information on associated changes in leaf gas exchange and other leaf traits can be valuable for modelling biogeochemical fluxes under altered land-use patterns. We studied in situ photosynthetic parameters and stomatal conductance for water vapour in eight abundant tree species of young secondary forest and eight tree species of natural old-growth forest in Central Sulawesi, Indonesia. In sun leaves, the average maximal stomatal conductance (g _smax) in the secondary forest (SF) species was 2.1 times higher than in the old-growth forest (OGF) species. Species with a high g _smax reduced g _s sharply when vapour pressure deficit of the air increased, whereas species with a low g _smax were much less sensitive to air humidity. For area-based photosynthetic capacity (A _max-area), the SF species had a 2.3 times higher average than the OGF species. For both, g _smax and A _max-area the variation among species was higher in the OGF than in the SF. When all tree species (n=16) are considered, species means of specific leaf area (SLA), leaf N concentration and leaf P concentration were significantly correlated with g _smax and A _max-area. The strong correlation between A _max-area and foliar P (r ²=0.8) is remarkable as the alluvial soils in the study region are rich in nutrients. If the eight OGF species are analysed separately, the only significant correlation was observed between SLA and mass-based A _max; in the SF species strong correlations were found between leaf size and A _max-area and g _smax. These results show that the conversion of old-growth forest to young secondary forest in Sulawesi significantly alters tree leaf gas exchange characteristics and that chemical and structural leaf traits can be used for the prediction of these changes. The best correlations between leaf gas exchange parameters and leaf traits were obtained by different traits in the SF species, the OGF species and the entire pool of studied species.     

Monitoring Lactobacillus plantarum in grass silages with the aid of 16S rDNA-based quantitative real-time PCR assays

Ensiling plant material with the aid of lactic acid bacteria (LAB) is a common agricultural practice for conserving forages independently of the time point of harvest. Despite ensiling being a natural process, it can be improved by the treatment of the harvested forage with starter cultures before storage. Within this context, Lactobacillus plantarum (L. plantarum) is the most frequently used LAB in commercially available starter cultures. In order to enable the monitoring of the population dynamics of L. plantarum in silage, methods for species-specific detection based on the 16S ribosomal DNA (rDNA) sequence were developed by applying a quantitative real-time polymerase chain reaction (QRT-PCR) approach. The QRT-PCR assay was also applied to estimate the development of the L. plantarum population within experimental grass silages. In addition, a multiplex QRT-PCR assay was developed to estimate the amount of L. plantarum 16S rDNA in relation to total bacterial 16S rDNA. This multiplex QRT-PCR assay was applied to monitor the influence of different silage additives on the L. plantarum population.     

Inorganic soil nitrogen under grassland plant communities of different species composition and diversity

We measured aboveground plant biomass and soil inorganic nitrogen pools in a biodiversity experiment in northern Sweden, with plant species richness ranging from 1 to 12 species. In general, biomass increased and nitrate pools decreased with increasing species richness. Transgressive overyielding of mixed plant communities compared to the most productive of the corresponding monocultures occurred in communities with and without legumes. N₂-fixing legumes had a fertilizing function, while non-legumes had a N retaining function. Plant communities with only legumes had a positive correlation between biomass and soil nitrate content, whereas in plant communities without legumes they were negatively correlated. Both nitrate and ammonium soil pools in mixed non-legume communities were approximately equal to the lowest observed in the corresponding monocultures. In mixed legume/non-legume communities, no correlation was found for soil nitrate with either biomass or legume biomass as percentage of total biomass. The idea of complementarity among species in nitrogen acquisition was supported in both pure non-legume and mixed non-legume/legume communities. In the latter, however, facilitation through increased nitrogen availability and retention, was probably dominating. Our results suggest that diversity effects on biomass and soil N pools through resource use complementarity depend on the functional traits of species, especially N₂ fixation or high productivity.     

Water channels in Chara corallina

Water relations parameters ofChara corallina inter-nodes weremeasured using the single cell pressure probe. The effect ofmercurials, which are recognized as non-specific water channelinhibitors, was examined. HgCl₂ concentrations greater than5 mmol m^–3 were found to inhibit hydraulic conductivity{Lp) close to 90%, whereas pCMPS was found to have no effecton Lp. The activation energy of water flow was increased significantlyfrom 21.0 kJ mol^–1 to 45.6 kJ mol^–1, following theapplication of HgCl₂. These results are in accordance with evidencefor Hg²⁺sensitive water channels in the plasma membrane of charophytes(Henzler and Steudle, 1995; Tazawa et al., 1996). The metaboliceffects must, however, be considered in view of the rapid inhibitionof respiration and the depolarization of the membrane potentialwith HgCl₂ concentrations lower than those found to affect Lp.It was possible to measure simultaneously water relations andmembrane PD, in order to examine the contribution of potassiumchannels to Lp. Cells were induced into a K⁺ permeable state.The K⁺ channels, assumed to be open, were subsequently blockedby various blockers. No significant difference in Lp was foundfor any of these treatments. Finally, the permeability of C.corallina membranes to ethanol was examined. HgCl₂ was foundto cause a decrease in reflection coefficient, coinciding witha decrease in Lp, but there was no change in the ethanol permeabilitycoefficient. This has been interpreted in terms of both thefrictional model and composite model of non-electrolyte membranetransport. Key words: Water channels, Chara, hydraulic, conductivity, membrane transport models, reflection coefficient     

Cholesterol diet leads to attenuation of ischemic preconditioning-induced cardiac protection: the role of connexin 43

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43.     

Expression of δ-toxin by Staphylococcus aureus mediates escape from phago-endosomes of human epithelial and endothelial cells in the presence of β-toxin

Staphylococcus aureus is able to invade non-professional phagocytes by interaction of staphylococcal adhesins with extracellular proteins of mammalian cells and eventually resides in acidified phago-endosomes. Some staphylococcal strains have been shown to subsequently escape from this compartment. A functional agr quorum-sensing system is needed for phagosomal escape. However, the nature of this agr dependency as well as the toxins involved in disruption of the phagosomal membrane are unknown. Using a novel technique to detect vesicular escape of S. aureus, we identified staphylococcal virulence factors involved in phagosomal escape. Here we show that a synergistic activity of the cytolytic peptide, staphylococcal δ-toxin and the sphingomyelinase β-toxin enable the phagosomal escape of staphylococci in human epithelial as well as in endothelial cells. The agr dependency of this process can be directly explained by the location of the structural gene for δ-toxin within the agr effector RNAIII.