Fluorescence microscopy investigations of ligand propagation and accessibility under adherent cells

Fluorescence microscopy methods including total internal reflection fluorescence and confocal laser scanning microscopy have played a major role in modern cell biology research by permitting imaging of fluorescently tagged macromolecules in living cells. These methods are often used to examine the initial events in signal transduction, which involve interactions occurring between membrane receptors and ligands such as antibodies and growth factors. Most quantitative biophysical applications using these fluorescence imaging methods, including ligand binding assays, are based on the assumption that the fluorophore label of interest has equal access to all areas of the membrane on the cell. Our findings suggest that there is limited accessibility of fluorophores (25±2%)(-) under the basal membrane of adherent CHO-K1 cells expressing epidermal growth factor receptor plated on a bare glass in standard two-dimensional tissue cultures. The authors present a detailed study of the extent to which a small fluorescent dye molecule (Alexa 647) is able to propagate under the basal membrane of cells plated on a variety of biologically compatible substrates: fibronectin, bovine serum albumin, poly-d-lysine, collagen I, collagen IV, Geltrex?, and fibronectin such as binding polymer. For nonspecific dye propagation the best overall accessibility was achieved using a thin layer preparation of a commercially available basement membrane matrix, Geltrex? (67±8%). Coupling of a specific high affinity ligand (epidermal growth factor) to the dye did result in a moderate increase in propagation for most substrates examined. Despite the overall increase in propagation for most substrates (60%-80%), large areas under the central regions of the adherent cells still remained inaccessible to the fluorescently labeled ligand. More importantly, the presence of the specific ligand did not result in consistent increase in ligand propagation. Taken together these results suggest that the reduced accessibility is not exclusively due to steric effects, and the chemistry of both the ligand and the substrate may be important when working under conditions of reduced dimensionality.     

The impact of staphylococcal mastitis on the level of milk IL-6, lysozyme and nitric oxide

Mammary gland secretions derived from secretory cows infected with coagulase +ve Staphylococcus spp. was examined for the expression of IL-6, production of lysozyme and NO(x). The examined cows reflected 25 cases of subclinical mastitis and 15 cases of clinically mastitic animals. The IL-6 concentration in the subclinical animals was significantly higher (30.8 ng/ml) than the clinically manifested animals (18.0 ng/ml) and the normal cows (5.2n g/ml). On the other hand the level of lysozyme although significantly higher than the normal cows (6.9 microg/ml) yet its level in the subclinical animals (11.2 microg/ml) was lower than that estimated in the clinical animals (15.6 microg/ml). Similarly, the level of NO(x) in the normal animals was found to be 5.6 microM/ml to increase to 6.2 microM/ml in the subclinical mastitic animals and to significantly increase further to 11.5 microM/ml in the clinically affected cows. These results suggest the promising use of whey IL-6, lysozyme or/and NO concentration variabilities as prognostic parameters on the degree of the commencement of mastitis in cows.     

Microbial biomass and soil organic carbon accumulation on a former lakebed near Novosibirsk,Russia

Shrinking of Lake Chany in W Siberia exposed 10,000 km² of former lakebed. On this new land we studied a chronosequence of young soils. The fraction of microbial C in total organic C had stabilized at 3% after 30 to 40 y. Concentration of organic C increased within the first 50 to 100 y to 4–5% in the first 10 cm, and to 2–3% at 10–20 cm depth. The amount of sequestered C is equivalent to about 2.5 d of current anthropogenic CO₂ emissions.     

Evolution of coral pigments recreated

In proteins homologous to the green fluorescent protein (GFP), formation of red fluorescence requires three autocatalytic steps, whereas only two are needed for green fluorescence. Multiple red/green color diversification events in the GFP superfamily may reflect convergent evolution of the more complex three-step pathway. In the great star coral Montastraea cavernosa, a recreated common ancestor of green and red proteins turned out to be green, indicating that in this case red proteins evolved their color independently from most other homologous red proteins. Furthermore, red color appears to have evolved gradually by small incremental transitions.     

Soil mesofauna and diversity of vegetation: Collembola in pristine taiga forests (Pechora-Ilych Biosphere Reserve,Russia)

Unique 600-year-old tall herb taiga forests are located in the European North-East of Russia at the foothills of the Middle Urals and are characterized by extremely diverse and vertically differentiated vegetation. This study addresses how vegetation parameters such as net primary productivity and diversity influence the community structure of Collembola in fir forests of different ages. Sample plots were arranged along diversity and biomass gradients of vegetation: Vaccinium-moss, short herb-moss, and nitrophilous tall herb fir–spruce forests. Plants and collembolans were compared with respect to their species richness, abundance, species structure and traits. The number of species of vascular plants, mosses and liverworts along the studied gradient increased by about double, whereas the species richness of collembolans did not differ significantly. The biomass of vascular plants increased, whereas that of mosses declined, and the abundance of collembolans remained approximately consistent. Despite sharp differences in ground vegetation, in all phytocenoses the dominant Collembola species were widespread. However, the collembolan community of old-growth tall herb forests showed a trend of more complex structure reflected by greater evenness of abundances compared to younger ecosystems with less diverse ground vegetation. In conclusion, the springtail communities in forests that have developed spontaneously for over 600 years appeared to be no more complex than those in 200-year-old stands. Multi-species ground vegetation did not increase collembolan diversity and abundance indices. Thus, soil mesofauna (in this case, springtails) appeared to be relatively independent of long-term spontaneous development in vegetation in the studied area.