Hemorheological aspects of the endothelium-plasma interface.
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The capillary perfusion model according to Cazenave and co-workers was characterized by investigating the effects of protein precoating, perfusion time and shear rate on platelet deposition using 111Indium labelling of human platelets and scanning electron microscopy (SEM). Compared with uncoated polyethylene, platelet deposition was increased after precoating with purified human von Willebrand factor, fibrinogen or fibronectin, and decreased by preadsorbed immunoglobulin G, albumin or whole plasma. Platelet aggregates were observed on immunoglobulin G-coated polyethylene, whereas all other surfaces showed single adherent platelets. Complete platelet spreading was only observed after precoating with fibronectin. The quantitative data concerning platelet deposition were evaluated by using the convective-diffusion theory. Our results indicate the applicability of this perfusion model for the in vitro testing of biomaterials.
The migration inhibiton test in agarose as described by Clausen (Acta Allergol 26:56, 1971) was modified into a statistically designed assay comprising a laboratory standard in each assay, and using buffy coat cells or peripheral polymorphonuclear leukocytes from pigs as target cells. The precision of the assay was improved by combining cells from several pigs. Fifty-5000 microliters of lymphokine-containing test fluid could be used in the assay provided that this fluid contains the essential nutrients. In our hands, the capillary tests was unsuitable for a quantitative assessment of migration inhibition activity.
The rheological characteristics of bovine amniotic fluid have been studied at different shear rates. The viscosity of bovine amniotic fluid at 20 degrees C was found to increase with time at a constant low shear rate during the measurement. Additionally, the viscosity was observed to decrease with increasing shear rate, indicating that a shear thinning behaviour of the fluid was occurring. The log-log plot of shear stress versus shear rate yielded a straight line consistent with non-Newtonian behaviour of the fluid and characteristic of pseudoplastic liquids. The data of shear stress versus shear rate could be represented by a power law model. The treatment of amniotic fluid with cetylpyridinium chloride (CPC) resulted in the precipitation of a mixture of components, including complex sulphated polysaccharides and extracellular proteoglycans, with the viscosity of the resulting liquid similar to that of water at 20 degrees C. In addition, the viscosity of the CPC-pretreated fluid did not increase with time at constant shear rate and remained constant with the increase in shear rate. The apparent increase in viscosity with time and the shear thinning behaviour of the amniotic fluid can thus be attributed to pseudoplastic liquid behaviour associated with the presence of structurally complex polysaccharides and extracellular proteoglycans. The implications of this fluid viscosity behaviour are discussed in terms of their impact on the operation of packed or expanded (fluidized) chromatographic bed systems when amniotic fluid biofeedstocks are used as a source of commercially important proteins.
UNLABELLED: The possibility of performing bioconversions under an electric field is here reported. A system is described by which the enzyme is trapped by an isoelectric mechanism between two zwitterionic membranes having pI values encompassing the isoelectric point of the enzyme. The enzyme is loaded into a multicompartment electrolyzer and kept operating under an electric field, which will continuously harvest the reaction product. Since, under focusing conditions, all buffering ions will vacate the reaction chamber at steady state, the buffering ion is trapped into the enzyme chamber by using amphoteric buffers co-isoelectric with the enzyme. As an example of such "isoelectrically immobilized' reactor, the enzyme beta-hydroxysteroid dehydrogenase is blocked into an isoelectric trap delimited by a pI 8.0 and a pI 6.5 membranes. 100 mM histidine (pI 7.47) is co-immobilized by the same isoelectric mechanism into the enzyme chamber. The dehydrocholic acid substrate (3,7,12-trioxo-5 beta-cholanoic acid) and reduced co-factor (NADH) are continuously infused into the enzyme chamber and the product (3 beta-hydroxy-7,12-dioxo- 5 beta-cholanoic acid, a compound of pharmaceutical interest) and the oxidized co-factor (NAD+) collected, separately, into the two neighbouring chambers at the anodic side. ADVANTAGES: in a soluble form, the enzyme maintains the reaction kinetics of the free soluble form. Additionally, the reaction product and exhausted co-factor can be recovered by electrophoretic transport.
The Immobiline (weak acrylamido acids and bases) chemistry has been applied to the covalent attachment of a positively (or, if needed, negatively) charged layer onto the inner surface of the silica wall. In particular, the following basic Immobilines have been used: pK 6.2, pK 7.0, pK 8.5 and pK 9.3. In order to avoid pK changes, the charged Immobilines are mixed with neutral acrylamido derivatives (in particular the highly resistant and hydrophilic N-acryloyl aminoethoxyethanol) so as to form a co-polymer having a 1:5 molar ratio (charged to neutral). The mu(eo) vs. pH curves have a slope opposite to that of a naked capillary and fan out on the pH scale following the titration curves of the different weak bases. Such chemistry allows the covalent attachment of charged species having known pK values and offering controlled charged densities on the wall. However, with the atomic force microscope, it is found that such soft coatings (whether charged or neutral) do not seem to provide complete coverage of the surface: naked patches of fused silica are found interdispersed among the polymer-coated ones. A good solution is a hybrid bonded and dynamic coating, obtained by adding short chain linear polyacrylamides to the background electrolyte. Good separations of polycations [poly(L-histidine)] and of histones are reported up to pH 5.7.
This report represents an investigation into the nature of apoptosis in hybridoma cultures and its significance to their utilization in biotechnology. To this end DNA fragmentation and capillary electrophoresis of genomic DNA was studied during the culture of two hybridoma cell lines. This indicated that the phenomenon of apoptosis was always present even under normal culture conditions. Two DNA fragments not associated with the typical DNA fragmentation ladder were identified in the two hybridoma cultures: a previously unreported DNA fragment of about 100 bp and a large fragment which may correspond to one reported in the literature (Walker et al., 1993). The small fragment was identified as soon as the early exponential growth phase of culture, while the large fragment appeared only in the latter part of the growth curve when there was marked DNA fragmentation. In addition we present evidence that aurintricarboxylic acid, which inhibits apoptosis in neural cells, permits this process in hybridoma cells at levels below 100 microM. This unusual predisposition of hybridoma cultures to undergo apoptosis and their response to inhibitor of apoptosis may have important implications for approaches to the culture of hybridomas and their utilization for monoclonal production.
The question is posed: Why does a living cell react to the absence of gravity? What sensors may it have? Does it note pressure, sedimentation, convection, or other parameters? If somewhere in a liquid volume sodium ions are replaced by potassium ions, the density of the liquid changes locally: the heavier regions sink, the lighter regions rise. This may contribute to species transport, to the metabolism. Under microgravity this mechanism is strongly reduced. On the other hand, other reasons for convection like thermal and solutal interface convection are left. Do they affect species transport? Another important effect of gravity is the hydrostatic pressure. On the macroscopic side, the pressure between our head and feet changes by 0.35 atmospheres. On the microscopic level the hydrostatic pressure on the upper half of a cell membrane is lower than on the lower half. This, by affecting the ion transport through the membrane, may change the surrounding electric potential. It has been suggested to be one of the reasons for graviperception. Following the discussion of these and other effects possibly important in life sciences in space, an order of magnitude analysis of the residual accelerations tolerable during experiments in materials sciences is outlined. In the field of life sciences only rough estimates are available at present.