Magnetically Self-Assembled Colloidal Three-Dimensional Structures as Cell Growth Scaffold.

Understanding the chemical and physical conditions for cell growth is important from biological and medical aspects. Many tissues and cell types (e.g., epithelial cells and neurons) naturally grow on surfaces that span in three-dimensions and offer structural or mechanical support. The scaffold surface has to promote adhesion and cell proliferation as well as support their weight and retain its structural integrity. Here, we present a flexible method that uses self-assembly of micrometer superparamagnetic particles to produce appropriate scaffold surfaces with controllable general appearance in three dimensions, such as oriented membranes, branched structure, or void network. As a proof of principle, the Chinese hamster ovary epithelial cell line was successfully grown for several days on inclined membranes. Robustness of the oriented membrane architecture was probed with optical tweezers. We measured the magnetic force holding one particle in a self-assembled upright hexagonal sheet and modeled it as a sum of pair interaction forces between spatially arrested static dipoles.

New synthetic routes for the preparation of ruthenium-1,10-phenanthroline complexes. Tests of cytotoxic and antibacterial activity of selected ruthenium complexes.

Three novel complexes have been prepared through reactions of precursor [(dmso)2H][trans-RuCl4(dmso-S)2] (P) and 1,10-phenanthroline (phen) at different conditions. Whereas the analogs of mer-[RuCl3(dmso-S)(phen)] (1) and [Ru(phen)3]Cl2·6CH3OH (3·6CH3OH) have already been prepared by other synthetic routes before, product (H3O)[RuCl4(phen)]·4H2O (2·4H2O) is unprecedented. In the latter, isolated from highly acidic medium, one strongly bound dmso molecule in precursor P was substituted by chloride. Biological activity of 1 and previously isolated ruthenium-purine complexes ([mer-RuCl3(dmso-S)(acv)(CH3OH)] (4) (acv = acyclovir); [trans-RuCl4(dmso-S)(guaH)] (5) (guaH = protonated guanine)) was tested and compared. These data show that compounds 1, 4 and 5 are slightly cytotoxic against B-16 malignant melanoma cells but not against non-transformed V-79-379A cells. It seems that coordinated phen ligand increases the cytotoxicity of 1 in comparison to ruthenium precursor. The inability of tested compounds to induce lysis of bovine erythrocytes suggests that their cytotoxic effect is not due to the membrane damage.

Positioning of integrin ß1, caveolin-1 and focal adhesion kinase on the adhered membrane of spreading cells.

We have investigated the relationship between the spreading of anchorage-dependent cells and the surface-density distribution of plasma membrane adhesion proteins. The surface positioning and density of integrin ß1, caveolin-1 (cav-1), the phosphorylated caveolin-1 (p-cav-1) and the focal adhesion kinase (FAK) located on the adhering cell membrane (ACM) of HUVEC cells was studied. Imaging with TIRF microscopy was used, which enabled us to observe a few-nanometers-thin section of the cell above the plasma membrane in combination with image-based analyses. Integrin ß1 and cav-1 have spatial interdependence on the ACM. Cells treated with substances that act on cell spreading caused changes in the size of the ACM area, as well as a redistribution of several proteins under investigation. Changes to the ACM area correlated positively with those to the surface density of the cav-1. The high integrin ß1 and the low cav-1 surface density, and vice versa, following the treatments show that the presence of one of them not only spatially excludes, but also reduces, the occurrence of the other protein on the ACM, which indicates a regulative mechanism between integrin ß1 and cav-1.

Biological activities of ethanolic extracts from deep-sea Antarctic marine sponges.

We report on the screening of ethanolic extracts from 33 deep-sea Antarctic marine sponges for different biological activities. We monitored hemolysis, inhibition of acetylcholinesterase, cytotoxicity towards normal and transformed cells and growth inhibition of laboratory, commensal and clinically and ecologically relevant bacteria. The most prominent activities were associated with the extracts from sponges belonging to the genus Latrunculia, which show all of these activities. While most of these activities are associated to already known secondary metabolites, the extremely strong acetylcholinesterase inhibitory potential appears to be related to a compound unknown to date. Extracts from Tetilla leptoderma, Bathydorus cf. spinosus, Xestospongia sp., Rossella sp., Rossella cf. racovitzae and Halichondria osculum were hemolytic, with the last two also showing moderate cytotoxic potential. The antibacterial tests showed significantly greater activities of the extracts of these Antarctic sponges towards ecologically relevant bacteria from sea water and from Arctic ice. This indicates their ecological relevance for inhibition of bacterial microfouling.

Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells.

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V-79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye-exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin-probe MeFASL(10,3) (5-doxylpalmitoyl-methylester), which partitions mainly in cell membranes and the hydrophilic spin-probe TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin-probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin-treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.

Ostreolysin affects rat aorta ring tension and endothelial cell viability in vitro.

The effects of ostreolysin, a cardiotoxic cytolysin produced by the edible oyster mushroom (Pleurotus ostreatus), were studied on tension development in isolated rat aortic ring. Its cytotoxic effects on human umbilical vein endothelial cells and Chinese hamster lung fibroblasts were also studied. Ostreolysin induced a concentration-dependent increase in aortic ring tension in the range from 5 to 30 microg/ml, and was cytotoxic to both cell lines. These effects could contribute significantly to its previously observed cardiotoxicity.

Isolation and primary culture of Necturus maculosus (Amphibia: Urodela) hepatocytes.

In order to evaluate their suitability for physiological and ecotoxicological studies, hepatocytes were isolated from the common mudpuppy (Necturus maculosus) using a two-step collagenase perfusion. Hepatocytes in primary culture were investigated for 14 d using light and electron microscopy and biochemical analyses. A typical perfusion yielded 1.7 x 10(5) viable hepatocytes per gram body weight with an average viability of 86 +/- 5%. The majority of isolated cells remained in suspension and formed aggregates. The viability of hepatocytes in primary culture was dependent on a fetal calf serum (FCS) concentration and incubation temperature. Viability was best at 8 degrees C in Leibovitz L-15 medium supplemented with 5% FCS. The ultrastructural characteristics of freshly isolated hepatocytes resembled those of N. maculosus hepatocytes in vivo. Whereas hepatocyte viability remained relatively stable (around 80%) up to 14 d in culture, electron microscopic analyses revealed changes at ultrastructural level. The majority of hepatocytes retained similar structural characteristics to those in vivo up to 4 d. Loss of cellular polarity, fractionation of rough endoplasmic reticulum, formation of autophagosomes, and successive exhaustion of cellular glycogen deposits were observed with increased time in culture. Functional integrity, as estimated by tyrosine aminotransferase induction, decreased during the culture period. Ultrastructural and biochemical analyses indicate the need for further improvement of culture conditions. Nevertheless, isolated hepatocytes in primary culture for up to 4 d can be recommended as a model for physiological and toxicological studies in lower vertebrates.

The influence of the successive depolymerization and polymerization of cytoskeleton components on the fibroblast shapes.

Monitoring the influence of the cytoskeleton polymers on the shape of fibroblasts, performing the experiments of repeated degradation and polymerization of microtubules and microfilaments, we found out that the presence of microtubules is necessary in order to regenerate the proper functional structure of microfilaments, and vice versa.

Ostreolysin, a pore-forming protein from the oyster mushroom, interacts specifically with membrane cholesterol-rich lipid domains.

Ostreolysin, a 15 kDa pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their cholesterol contents and with the occurrence of ostreolysin in the cells detergent resistant membranes. Moreover, ostreolysin binds to supported monolayers and efficiently permeabilizes sonicated lipid vesicles, only if cholesterol is combined with either sphingomyelin or dipalmitoylphosphatidylcholine. Addition of mono- or di-unsaturated phosphatidylcholine to the cholesterol/sphingomyelin vesicles dramatically reduces the ostreolysin's activity. It appears that the protein recognizes specifically a cholesterol-rich lipid phase, probably the liquid-ordered phase.

Effects of periodontal dressings on fibroblasts and gingival wound healing in dogs.

In the present study the effects of different commercially available periodontal dressings (Peripac, Barricaid, Fittydent, Reso-Pack and Myzotect-tincture) on fibroblast (V-79-379A) proliferation and survival were tested in vitro. Barricaid, Fittydent and Reso-Pack periodontal dressings have only small inhibitory effects on cell proliferation (83.3 +/- 9%, 71.6 +/- 8.7% and 87.3 +/- 4.5% of control after 48 h, respectively) in comparison with the great inhibitory effect of Myzotect-tincture (2.9 +/- 0.1%) and Peripac (33.7 +/- 11.4%) (p < 0.001). Barricaid was the only dressing where 41% of cells survived after exposure, while the other four dressings killed all the cells in 6 days. In addition, the healing of artificially created gingival wounds covered by Barricaid and Reso-Pack was followed for 7 days in 12 Beagle dogs. Histological evaluation of gingival tissue demonstrated that wounds covered by Reso-Pack showed the best epithelisation and vascularity and the least inflammatory reaction in first 4 days. Later the observed parameters were similar with those of wounds covered by Barricaid or without pack. The present results indicate that Peripac periodontal dressing and Myzotect-tincture showed the highest cytotoxicity to fibroblasts in vitro. From the histological observations in Beagle dogs Reso-Pack has been found to be the most suitable dressing, followed by Barricaid.

Biological activities of organic extracts of four Aureobasidium pullulans varieties isolated from extreme marine and terrestrial habitats.

We report on the screening for biological activities of organic extracts from seven strains that represent four varieties of the fungus Aureobasidium pullulans, that is A. pullulans var. melanogenum, A. pullulans var. pullulans, A. pullulans var. subglaciale and A. pullulans var. namibiae. We monitored haemolysis, cytotoxicity, antioxidant capacity and growth inhibition against three bacterial species. The haemolytic activity of A. pullulans var. pullulans EXF-150 strain was due to five different haemolytically active fractions. Extracts from all of the other varieties contained at least one haemolytically active fraction. Short-term exposure of cell lines to these haemolytically active organic extracts resulted in more than 95% cytotoxicity. Strong antioxidant capacity, corresponding to 163.88 µg ascorbic acid equivalent per gram of total solid, was measured in the organic extract of the strain EXF-3382, obtained from A. pullulans var. melanogenum, isolated from the deep sea. Organic extracts from selected varieties of A. pullulans exhibited weak antibacterial activities.

Fatty acid composition of common barbel (Barbus barbus) roe and evaluation of its haemolytic and cytotoxic activities.

Eggs of the common barbel (Barbus barbus) cause intoxication in humans after ingestion. In this work, the chemical composition of the haemolytically active fraction from methanolic barbel roe extract was analyzed. Compounds showing haemolytic activity and cytotoxicity towards normal and transformed cell lines were isolated and identified as polyunsaturated fatty acids, using online liquid chromatography-electrospray ionization mass spectrometry through tandem fragmentation experiments (HPLC-MS/MS). Arachidonic acid (C20:4), docosahexaenoic acid (C22:6) and eicosapentaenoic acid (C20:5) proved to be the three most abundant members of a complex series of free fatty acids ranging from C14:0 to C24:5.

Time lapse monitoring of CaCo-2 cell shapes and shape dependence of the distribution of integrin ß1 and F-actin on their basal membrane.

CaCo-2 cell line is a model system for cell differentiation. For the effective use of CaCo-2 cells, it is important to understand how their growth depends on environmental conditions. The authors grew them on laminin-1, fibronectin, and collagen-1 adsorbed to glass and polystyrene. The time lapse technique was applied to follow their growth and shape changes for 21.5 h post seeding. The results upgraded the auhtors' previous findings about the series of consecutive shape changes that occur post seeding. Most cells were initially rounded and then they changed shape in two directions. A smaller fraction of cells, which attained cumulus shapes, eventually detached and drifted away. Other cells attained a semispread, transient shape, which was followed by a fully spread shape that was dominant on all protein-coated surfaces. The average time over which cells changed their shape type was different on different surfaces. It was longer on protein-coated glass surfaces than on protein-coated polystyrene surfaces. On collagen-1-coated surfaces, cells spread in the shortest time. Different cell shape types exhibited different spatial distributions of integrin ß1, F-actin, and focal adhesions.

Redistribution of cytosolic FGFR1 after induced migration of urothelial cells in culture.

The distribution of cytosolic fibroblast growth factor receptor 1 (FGFR1) was studied in correlation to cell migration in urothelial cell line g/G. Cell motility was analysed with a new method using consecutive series of photographs of cells relocated on CELLocate coverslips and with image analysis software. The results confirmed that FGF1 stimulated cell motility only when cells were grown on collagen I coating. During the transition from sessile to motile cell phenotype a complete redistribution of cytosolic FGFR1 was revealed. In sessile cells, FGFR1 had a filamentous distribution and its location matched cytokeratin 7. In cells of the migrating phenotype, the distribution of FGFR1 was diffuse, mainly located in cytosol. Our data reveal that the location of cytosolic FGFR1 depends on the motile characteristics of the cell. The results also indicate that attachment of cells to collagen I is crucial for the induction of urothelial cell motility with FGF1.