Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion

The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of alpha5 integrin also occurs in the same stiffness range, but exogenous expression of alpha5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands.     

Effects of growth temperature on the adhesion ofPseudomonas aeruginosa ATCC 27853 to polystyrene

The aim of this work has been to analyse the effects of temperature on polystyrene adhesion ofPseudomonas aeruginosa ATCC 27853. The bacterial adhesion (expressed as percentage of hydrophobicity) has been measured during the cultivation of this strain at different temperature of growth (15, 30 and 47°C). Data obtained showed that an increase in temperature is a factor that increasing the virulence of the strain in terms of adhesion to polystyrene surfaces. This kind of experiments surely brings important information concerning the prevention of nosocomial infection.     

Ubiquitin links to cytoskeletal dynamics, cell adhesion and migration

Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin. The protein ubiquitylation machinery is remarkably complex, comprising more than 15 Ubls (ubiquitin-like proteins) and several hundreds of ubiquitin-conjugating enzymes. Ubiquitin is best known for its role as a tag that induces protein destruction either by the proteasome or through targeting to lysosomes. However, addition of one or more Ubls also affects vesicular traffic, protein-protein interactions and signal transduction. It is by now well established that ubiquitylation is a component of most, if not all, cellular signalling pathways. Owing to its abundance in controlling cellular functions, ubiquitylation is also of key relevance to human pathologies, including cancer and inflammation. In the present review, we focus on its role in the control of cell adhesion, polarity and directional migration. It will become clear that protein modification by Ubls occurs at every level from the receptors at the plasma membrane down to cytoskeletal components such as actin, with differential consequences for the pathway's final output. Since ubiquitylation is fast as well as reversible, it represents a bona fide signalling event, which is used to fine-tune a cell's responses to receptor agonists.     

Cell-to-cell adhesion of dissociated embryonic brain cells; the effects of metabolic inhibitors and temperature

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and rotated for 120 min. The area and density of of the adhesive complexes formed were registered using the method described previously. The adhesiveness of dissociated embryonic brain cells (measured during the 120 min of rotation) was diminished in the presence of inhibitors of protein synthesis (puromycin, cycloheximide and inhibition of mRNA synthesis actinomycin D). The inhibition was, however, not distinct, because 1 microgram/ml of cycloheximide and actinomycin was without any significant effect, and the degree of inhibition evoked by 10 micrograms/ml and 25 micrograms/ml of puromycin bordered on significance. However, protein synthesis inhibitors in long-term aggregation experiments had a pronounced inhibitory effect and/or induced destruction of the aggregates. Metabolic inhibitors (KCN and NaN3) caused an inhibition at the lowest level of significance (p less than 0.05) 10(-3) mol/l KCN reduced the final adhesive product significantly. Cells rotated at room temperature and at +5 degrees C adhere to the same extent as in control experiments (37 degrees C). The adhesion was significantly inhibited at +60 degrees C and also after freezing at -80 degrees C with subsequent thawing. The adhesion of cells exposed for 30 min to between +80 degrees C and 100 degrees C was completely abolished. The process of embryonic brain cell adhesion requires a low energy supply, and is relatively independent of biosynthetic processes and of temperature changes between +5 degrees C and +50 degrees C.     

The adhesion of BHK and PyBHK cells to the substratum

The adhesion of BHK 21/13 cells and their polyoma-transformed derivatives was studied by detaching them from plastic dishes with EGTA. The PyBHK cells were less adhesive, and the possibility that cyclic nucleotides might play a role in cell adhesion was examined. Both cAMP and cGMP increase cell adhesion through a mechanism involving microtubules, but glucocorticoids act to increase cell adhesion through an independent mechanism.We also examined the role of membrane fluidity in cell adhesion, and the results are discussed in terms of a general model for cell adhesion and locomotion.     

Effects of temperature, salinity and sex on the basal metabolic rate of the estuarine copepod Pseudodiaptomus hessei

Respiratory responses of Pseudodiaptomus hessei measured underdifferent conditions show an exponential increase in metabolicrate with temperature, but no changes with salinity. All metabolicdemands of this species are met through consumption of microalgaeduring the closed (winter) phase, but not during the open (summer)phase of the estuary.     

Mobility and cytoskeletal interactions of cell adhesion receptors.

Clustering of cell adhesion receptors and their interactions with the cytoskeleton are key events in the formation and function of cell adhesion structures. On the free cell surface, cadherin molecules interact with the cytoskeleton/membrane skeleton by being bound or corralled, and such interactions are greatly enhanced by the formation of cadherin oligomers. Corralled cadherin molecules undergo hop diffusion from one compartment to an adjacent one (membrane skeleton fence model), which prompts the initial formation of small adhesion clusters at cell-cell contact sites, but larger-scale assemblies of cadherin and actin filaments might require a further co-ordinated recruitment of these molecules.     

Effect of pH,temperature, and growth conditions on the adhesion of a gliding bacterium and three nongliding bacteria to polystyrene

The effect of growth rate, growth phase, pH, and temperature on the permanent adhesion of a glidingFlexibacter sp. and three nongliding bacteria,Pseudomonas fluorescens, Enterobacter cloacae, andChromobacterium sp., to polystyrene substrata was investigated. The permanent adhesion of the flexibacter appeared to be related to growth, as levels of adhesion increased with increased growth rate in continuous culture and declined rapidly with death phase in batch culture. With the three nongliding bacteria, there was no relationship between growth rate and levels of permanent adhesion. The permanent adhesion of the nongliding bacteria was maximum between pH 5.5 and pH 7 and between 20 and 30°C, whereas the adhesion of the flexibacter progressively decreased with increasing temperature and pH. The effect of different nutrient conditions on the gliding motility of the flexibacter across agar was also investigated. Gliding motility was inhibited by increased nutrient concentration and was affected by carbon source. Inhibition appeared to be related to the accumulation of a viscous exopolymer. It is proposed that the differences in the permanent adhesion of the gliding and nongliding bacteria may be related to their adaptation to different ecological niches.     

Modification of the calcium-independent mechanism of cell adhesion in transformed BHK cells

The calcium-independent mechanism of cell adhesion was studied in normal and polyoma virus-transformed BHK cells. The degree of Ca2+-independent adhesion was greatly reduced in pyBHK cells, whereas CA2+-dependent adhesion occurred to the same degree as in BHK cells. This decrease was shown not to be caused by simple masking of the adhesion sites or by their altered sensitivity to trypsin. Adhesion-blocking antibodies were used to identify molecules responsible for Ca2+-independent adhesion. The antibodies precipitated surface molecules specific for adhesion-competent cells. These have tentatively been named CIDSBHK and CIDSpyBHK. Both were glycoproteins with respective apparent molecular weights of 120K and 125K. CIDSpyBHK incorporated 3H-glucosamine more than CIDSBHK did. Possible modification of the Ca2+-independent adhesion mechanism in pyBHK cells is discussed.     

Effects of altered ambient temperature on metabolic rate during CO2 inhalation

The purpose of this study was to determine if the changes in O2 consumption (VO2) during CO2 inhalation could in part be due to stimulation of thermogenesis for homeothermy. Twelve ponies were exposed for 30-min periods to inspired CO2 (PIco2) levels of less than 0.7, 14, 28, and 42 Torr during the winter at 5 (neutral) and 23 degrees C ambient temperatures (TA) and during the summer at 21 (neutral TA), 30, and 12 degrees C. Elevating TA in both seasons resulted in an increased pulmonary ventilation (VE) and breathing frequency (f) (P less than 0.01) but no significant increase in VO2 (P greater than 0.05). Decreasing TA in the summer resulted in a decrease in VE and f (P less than 0.01) but no significant change in VO2 (P greater than 0.05). At neutral TA in both seasons, VO2 increased progressively (P less than 0.05) as PIco2 was increased from 14 to 28 and 42 Torr. The increases in VO2 during CO2 inhalation were attenuated (P less than 0.05) at elevated TA and accentuated at the relatively cold TA in the summer (P less than 0.05). Respiratory heat loss (RHL) during CO2 inhalation was inversely related to TA. Above a threshold RHL of 2 cal X min-1 X m-2, metabolic heat production (MHP) increased 0.3 cal X min-1 X m-2 for each unit increase in RHL during CO2 inhalation at the neutral and elevated TA. However, during cold stress in the summer, the slope of the MHP-RHL relationship was 1.6, indicating an increased MHP response to RHL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamic analysis of growth temperature dependence in the adhesion of Candida parapsilosis to polystyrene

The purpose of this work was to study the adhesion to polystyrene of two Candida parapsilosis strains, grown at 22 and 37 degrees C, in terms of hydrophobicity, surface charge, and interaction free energy. Growth temperature changed the surface properties of microorganisms, yielding a good correlation between thermodynamic predictions and adhesion behavior.     

Gigantism, temperature and metabolic rate in terrestrial poikilotherms

The mechanisms dictating upper limits to animal body size are not well understood. We have analysed body length data for the largest representatives of 24 taxa of terrestrial poikilotherms from tropical, temperate and polar environments. We find that poikilothermic giants on land become two-three times shorter per each 10 degrees of decrease in ambient temperature. We quantify that this diminution of maximum body size accurately compensates the drop of metabolic rate dictated by lower temperature. This supports the idea that the upper limit to body size within each taxon can be set by a temperature-independent critical minimum value of mass-specific metabolic rate, a fall below which is not compatible with successful biological performance.     

Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines

Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa (cervical cancer), A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types.     

Effects of metabolic inhibitors on spontaneous and interferon-boosted human natural killer cell activity

Human natural killer (NK) cell activity can be augmented by pretreatment with partially purified preparations of human interferon (IF). Studies have now been performed to determine the metabolic processes required for and involved in spontaneous NK activity and augmentation of cytotoxicity. A 4-hr 51Cr release cellular cytotoxicity assay was used to measure the NK activity, and peripheral blood leukocyte cells (PBL) were treated with: a) x-ray or mitomycin C; b) actinomycin D; or c) emetine, cycloheximide, pactamyhcin, or puromycin to assess the roles of DNA, RNA, and protein synthesis, respectively, in spontaneous NK activity and in boosting by IF. Prolonged incubation (18 hr) of PBL after blockage of synthesis of DNA almost completely abrogated NK activity; however, NK activity could be partially or totally restored to these populations by incubation of the effector cells for 1 hr at 37 degrees C with IF. Blockage of DNA synthesis for 1 hr had no effect on spontaneous NK activity or on boosting by IF. Inhibition of RNA synthesis also had no effect on spontaneous NK activity. Treatment of PBL with actinomycin before exposure to IF prevented boosting, but treatment with the RNA synthesis inhibitor after boosting with IF for 5 to 6 hr no longer had an appreciable effect on cytotoxicity. The effect of protein synthesis inhibitors on spontaneous NK activity was dependent on the inhibitor selected. Emetine and puromycin totally abrogated spontaneous NK activity at concentrations of inhibitor that blocked 3H-leucine incorporation 90% or more. In contrast, cycloheximide and pactamycin had only minimal effects on spontaneous NK activity but totally abrogated the boosting of IF.