The dualistic mechanisms of platelet adhesion to von Willebrand factor substrates

von Willebrand factor (vWf) which serves as a necessary factor for platelet adhesion to damaged vascular subendothelium can bind to the platelet surface via two distinct receptors. Ristocetin promotes the binding of vWf to platelet membrane glycoprotein lb, whereas platelet activation by thrombin supports binding to the glycoprotein IIb/IIIa complex. Platelet adhesion to vWf substrates mediated by these two mechanisms has been compared. Both mechanisms supported similar rates of adhesion to the substrates. Whereas adhesion via the ristocetin-dependent mechanism did not require divalent cations, adhesion mediated by the thrombin-dependent mechanism required the presence of divalent cations. Modification of vWf amino groups markedly impaired the ability of the protein to support ristocetin-dependent adhesion but did not alter its ability to support thrombin-enhanced adhesion. Reduction and carboxymethylation nearly abolished the ability of vWf to support adhesion via the ristocetin-dependent mechanism, but did not substantially impair its ability to support thrombin-enhanced adhesion. Short synthetic peptides containing the sequence Arg-Gly-Asp-Ser effectively inhibited thrombin-dependent platelet adhesion to vWf substrates but had no effect on ristocetin-dependent adhesion. Substrates composed of synthetic peptides containing the Arg-Gly-Asp-Ser sequence supported thrombin-dependent adhesion but did not support ristocetin-dependent adhesion. Scanning electron microscopic examination revealed that platelets adherent via the ristocetin-dependent mechanism almost uniformly adopted a flattened and fully spread appearance. In contrast, the thrombin-enhanced mechanism of adhesion supported only a limited degree of platelet spreading on the vWf substrate.     

On the role of calcium in adhesion of cells to solid substrates.

Experiments are described which show that while the presence of calcium in the medium is required for the cells to maintain their adhesion, it is not necessary for the initial attachment of 3T3 cells to solid substrates. Cells are detached by treatment with urea at 4 degrees C suggesting that adhesion may involve hydrogen bonding between the cell surface and the substratum. Although most of the cell-bound calcium is removed by trypsin, the detaching effect of trypsinisation can be inhibited at low temperature suggesting that ionic calcium bridges are probably not directly involved in retaining the cells on the surface. Cells are made totally insensitive to removal by trypsin by prior washing with lanthanum. Our findings suggest that the external role of calcium in cell adhesion is exerted indirectly. We conclude that the cell presents to the exterior at least two physiochemical classes of molecule. One class composed of hydrogen bond-forming adhesive material (possible proteins) and another class of anti-adhesive molecules (possibly glycoproteins). These two components are somehow separated in the formation of adhesive 'plaques' and this process is process is apparently uninfluenced by the calcium concentration in the medium. However, the maintenance of the localised zones of adhesion is aided by factors which prevent their disruption by the intrusion into them of anti-adhesive molecules diffusing from adjacent regions of the cell membrane. These factors include cooling below the transition temperature of the membrane lipids and lateral cross-linking of non-adhesive elements by calcium. By contrast, conditions which reduce the stability of the separation of adhesive and non-adhesive surface components would be expected to diminish the overall adhesiveness of cells to the substratum.     

Cellulose thin films: degree of cellulose ordering and its influence on adhesion

Adhesion measurements have been performed with thin cellulose films using continuum contact mechanics with application of the JKR theory. Three different cellulose surfaces were prepared, one crystalline and two surfaces with a lower degree of crystalline order. Adhesion between two cross-linked poly(dimethylsiloxane) (PDMS) caps, as well as the adhesion between PDMS and the various cellulose surfaces, was measured. The work of adhesion (from loading) was found to be similar for all three surfaces, and from contact angle measurement with methylene iodide it was concluded that dispersive interactions dominate. However, the adhesion hysteresis differed significantly, being larger for a less ordered cellulose surface and decreasing with increasing degree of crystalline order. This is suggested to be due to the surface groups' ability to orient themselves and participate in specific or nonspecific interactions, where a surface with a lower degree of crystalline order has a higher possibility for reorientation of the surface groups. The mobility of cellulose chains increases with water uptake, resulting in stronger adhesive joints. These films will hence allow for determination of the contributions of hydrogen bonding and inter-diffusion on the adhesion, determined from the unloading data, as the thermodynamic work of adhesion was found to be independent of the cellulose surface used.     

Laboratory studies on adhesion of microalgae to hard substrates

Adhesion of Chlorella vulgaris(chlorophyceae), Nitzschia amphibia(bacillariophceae) and Chroococcus minutus(cyanobacteria) to hydrophobic (perspex, titanium and stainless steel 316-L), hydrophilic (glass) and toxic (copper, aluminium brass and admiralty brass) substrata were studied in the laboratory. The influence of surface wettability, surface roughness, pH of the medium, culture age, culture density, cell viability and presence of organic and bacterial films on the adhesion of Nitzschia amphibia was also studied using titanium, stainless steel and glass surfaces. All three organisms attached more on titanium and stainless steel and less on copper and its alloys. The attachment varied significantly with respect to exposure time and different materials. The attachment was higher on rough surfaces when compared to smooth surfaces. Attachment was higher on pH 7 and above. The presence of organic film increased the attachment significantly when compared to control. The number of attached cells was found to be directly proportional to the culture density. Attachment by log phase cells was significantly higher when compared to stationary phase cells. Live cells attached more when compared to heat killed and formalin killed cells. Bacterial films of Pseudomonas putida increased the algal attachment significantly. %     

Electrochemical investigations of cholesterol enriched glassy carbon supported thin lipid films

The formation and study of stable cholesterol enriched thin lipid layers onto the surface of glassy carbon electrode is reported in this work. The method of formation relies on additional thinning of wetting films by electrostriction. Electrochemical techniques based on the concepts of impedance and voltammetry are used to explore the films' features. The impedance data reveal a substantial change of relaxation characteristics of the modified films. In this respect, opportunities for the evaluation of the films' stage based on the approximation with 'constant phase angle element' are discussed. The possible final structure of the films, as well as, their relevance for development of sensor elements are briefly viewed.     

Similarity of adhesion to phagocytosis: macrophage interaction with different substrates

Similarity between adhesion and phagocytosis is shown in the case of macrophages. Both adhesion of macrophages to polystyrene and phagocytosis of polystyrene particles (latex) do not depend much on the temperature and are not inhibited with iodoacetate. In contrast, both cell adhesion to adsorbed immunoglobulin G and phagocytosis of erythrocytes opsonized with immunoglobulin G, which are mediated with Fc-receptors, have some characteristic temperature dependence and are inhibited with iodoacetate. It may be concluded that it is the type of substrate, rather than its size, that determines mechanisms of adhesion and phagocytosis.     

The effect of dissolved organic carbon on bacterial adhesion to conditioning films adsorbed on glass from natural seawater collected during different seasons

Adhesion of three marine bacterial strains, i.e. Marinobacter hydrocarbonoclasticus, Psychrobacter sp. and Halomonas pacifica with different cell surface hydrophobicities was measured on glass in a stagnation point flow chamber. Prior to bacterial adhesion, the glass surface was conditioned for 1 h with natural seawater collected at different seasons in order to determine the effect of seawater composition on the conditioning film and bacterial adhesion to it. The presence of a conditioning film was demonstrated by an increase in water contact angle from 15 degrees on bare glass to 50 degrees on the conditioned glass, concurrent with an increase in the amount of adsorbed organic carbon and nitrogen, as measured by X-ray photoelectron spectroscopy. Multiple linear regression analysis on initial deposition rates, with as explanatory variables the temperature, salinity, pH and concentration of dissolved organic carbon (DOC) of the seawater at the time of collection, showed that the concentration of DOC was most strongly associated with the initial deposition rates of the three strains. Initial deposition rates of the two most hydrophilic strains to a conditioning film, increased with the concentration of DOC in the seawater, whereas the initial deposition rate of the most hydrophobic strain decreased with an increasing concentration of DOC.     

The relative ability of fungi from Sphagnum fuscum to decompose selected carbon substrates

Nine species from a suite of 55 microfungi isolated from living and decomposing Sphagnum fuscum were selected for studies of in vitro decomposition of tannic acid, cellulose, and starch. In vitro decomposition of S. fuscum plants and spruce wood chips was also examined. Oidiodendron maius and Oidiodendron scytaloides degraded tannic acid, giving a positive reaction for polyphenol oxidases. Most taxa degraded cellulose and starch via the synthesis of cellulases and amylase, respectively. Mass losses of spruce wood chips generally exceeded those of S. fuscum. A basidiomycete, similar to Bjerkandera adusta, caused the greatest mass losses in spruce wood chips (10.2%), while O. scytaloides caused the smallest mass losses (3.4%) after 8 weeks. For S. fuscum, Sordaria fimicola caused the greatest (5.1%) and Mucor hiemalis the smallest (0.1%) mass losses after 8 weeks. Filamentous microfungi have considerable potential to decompose a variety of carbon substrates of bryophilous residues in peatlands.     

The adhesion of pathogenic microflora on carbon sorbents

The adsorbing activity of granulated carbonic sorbents SKN and KAU, as well as their oxidated forms, containing protogenic carboxylic and phenolic groups with respect to Shigella flexneri, Salmonella typhimurium, Escherichia coli, Streptococcus aureus and Pseudomonas aeruginosa pathogenic strains has been studied. As shown in this study, the process of interaction between microorganisms and carbonic sorbents has two stages. At the first stage the main role is played by long-distance electrostatic forces and at the second stage, by Van der Waals short-distance forces, as well as bonds formed between cell structures and surface groupings of carbonaceous materials. In the mechanism of interaction between microbial cells and carbons the geometry of carbon surface plays an important role. KAU(0)-1 exhibits the highest degree of adhesion with respect to pathogenic bacteria.     

Substrate adhesion of rat hepatocytes: Mechanism of attachment to collagen substrates

Attachment of rat hepatocytes to collagen, which occurs without the aid of fibronectin, was found to be a time-dependent reaction characterized by an initial lag phase of 10–20 min before stable attachment bonds began to form. Increasing the density of molecules in the collagen substrates enhanced the rate of cell attachment. The hepatocytes attached essentially equally well to all the collagen types tested (types I, II, III, IV and V). The initial rate of cell attachment was more rapid to native collagen than to denatured collagen or α1(I) chains, apparently indicating different affinities of the cells for these substrates. However, if cells were incubated for 60 min or more, efficient attachment occurred to the α1(I) chain and to all cyanogen-bromide-treated peptides tested (α1-CB2, α1-CB3, α1-CB4, α1-CB5, α1-CB6A, α1-CB7, α1-CB8, α2-CB2, α2-CB3 and α2-CB4) but not to the aminopropeptide of type I procollagen. A low but significant degree of attachment also took place to substrates made of synthetic peptides with the collagen-like structures (Gly-Ala-Pro)_n, (Gly-Pro-Pro)_n and (Gly-Pro-Hyp)_n, whereas no attachment was observed to polyproline. We suggest that the cell-binding sites in collagen have a simple structure and occur in multiple copies along the collagen molecule. Addition of collagen in solution inhibited intial cell attachment, an effect that persisted longer on substrates made of α1(I) chain than on denatured collagen. The collected data are interpreted in terms of a model for cell-to-collagen adhesion where the formation of stable attachment bonds requires the binding of several low-affinity receptors, clustered at the site of adhesion, to collagen molecules in the substrate.     

Mutants of Escherichia coli K12 unable to grow on non-fermentable carbon substrates.

Among a number of mutants unable to utilize non-fermentable carbon substrates, scoring for membrane ATPase and for ATP-driven transhydrogenase activity permitted to distinguish two phenotypes: (A) mutants lacking ATPase and ATP-driven transhydrogenase; (B) one mutant with an ATPase which behaved according to several criteria as released into solution instead of being membrane bound, a.o it exhibited no ATP-driven transhydrogenase activity. All A and B mutants exhibited a common nutritional pattern. The ATPase-deficient group, when scored for ATPase-binding sites on its membrane particles revealed three different subgroups: (1) mutants having free ATPase-binding sites, (2) mutants with ATPase-binding sites made available by the procedure which releases ATPase from wild-type membrane, and (3) mutants with no detectable ATPase-binding sites. Membranes of the mutant B with unbound ATPase also exhibited a deficiency in ATPase-binding sites, but its soluble ATPase was also found unable to bind to ATPase-binding sites of wild type membranes. The double alteration, namely abnormal or inactive ATPase and absence of ATPase-binding sites on the membrane is compatible with a single mutational defect.     

The microstructure of isotropic vapor-deposited carbon films

The structure of thin, vapor-deposited carbon films was characterized by transmission electron microscopy and electron diffraction. Selected area electron diffraction showed very weak and broad peaks, indicating that these carbons contain extremely small crystallites whose dimension in the crystallographic c-direction is about 8 to 10 a. The observed diffraction bands are (h, k, 1 = 0) type reflections, which suggests that individual crystallites consist of graphitic layer planes stacked in parallel groups but with no order between atoms in adjacent planes (turbostratic). The carbon films exhibit no preferred orientation, indicating that the small crystallites are randomly oriented in the film and that the films are therefore isotropic. The measured density (1.8 g/cm3) and the structure of the vapor-deposited carbons are accordingly similar to those of low-temperature isotropic (LTI) pyrolytic carbons.     

Enthalpy changes associated with protein binding to thin films

Molecularly imprinted thin films consisting of proteins embedded in polymerised aminophenyl boronic acid have been made on glass supports. The protein contents of the films have been optimised to achieve a maximum energy of interaction between the film and the native template. The fabrication of the films and the subsequent removal from their surfaces of the imprint proteins has been shown to be a facile and easily reproduced process. The enthalpy changes associated with the rebinding of the films with their original templates (lysozyme and cytochrome c) and with non-native templates has been examined by micro-calorimetry. The results demonstrate that thin films can be successfully imprinted as shown by the significant reduction in the enthalpy (DeltaH) observed when the films were rebound with proteins other than the original templates. Additionally, it was shown that after binding, non-template proteins could be removed by washing and a greater enthalpy again observed when the films were rebound with the native protein compared to that which had been found with the non-native protein.     

Cell death in the thin films of bursting bubbles.

A sparged gas bubble floating at the liquid interface has a liquid film which drains and thins until the film spontaneously ruptures at a point. This causes rapid retraction of the film, forming a rim of collected fluid. This rim moves at a constant velocity of about 3 m/s and any cells in the bubble film are rapidly accelerated to this velocity in the moving rim. Half of the surface energy originally in the thin film is converted to kinetic energy of the rim, while the rest is dissipated in this rim. The rate of energy dissipation per mass of rim fluid is approximately 9000 m2/s3, which corresponds to a Kolmogorov eddy size of 3.2 microns in fully developed turbulence or a shear stress of 95 N/m2 in laminar flow. Either of these limiting cases presents an environment in which rapid cell death would be expected. Experiments with Sf-9 insect cells suggest that the cell concentration in these thin films is 0.6 times the bulk liquid concentration and that about 20% of these cells are killed when the film ruptures. An equation based on this mechanism accurately predicts the death rate.