Quantitative investigations of cell-bubble interactions using a foam fractionation technique

Previous work by the authors and others has shown that suspended animal cell damage in bioreactors is caused by cell-bubble interactions, regardless whether the bubbles are from bubble entrainment or direct gas sparging. As approach to measure the adsorptivity of animal cells to bubbles, a modified batch foam fractionation technique has been developed in this work and proven to be applicable. By using this technique, the number of cells adsorbed per unit bubble surface area and the adsorption coefficients have been measured to quantify hybridoma cell-bubble interactions, and the prevetive effects of serum and Pluronic F68 on these interactions. It was demonstrated quantitatively that the hybridoma cells adhere to bubbles spontaneously and significant numbers exist in the foam, and that both the serum and Pluronic F68 provide strong prevention to these cell-bubble interactions. The results obtained provide criteria for bioreactor operation and medium formulation to prevent cell-bubble interactions and cell damage in the culture processes.Abbreviations NBCS new born calf serum - SFM serum-free medium     

Model study of interactions of high-molecular dextran sulfate with lipid monolayers and foam films

The interaction of high-molecular dextran sulfate (DS-5000) with dimyristoylphosphatidylcholine (DMPC) monolayers and foam films (FF) at the air–water interface in the presence of Ca²⁺ and Na⁺ ions was studied. DS-5000 was added in monolayer films (MF) and in FF as monomer molecules and in liposomal form. When added in liposomal form in FF, DS-5000 decreased the stability of DMPC common black films (CBF), and no formation of Newton black films (NBF) was observed. However, when included as monomer molecules in FF, DS-5000 caused film thinning, and drastically decreased the expansion rate of the black spots and transition of thick films to NBF, thus avoiding formation of CBF. The above effects were observed in both gel and liquid-crystalline phase states of DMPC in the presence of Ca²⁺ ions only, and not in the presence of Na⁺ ions. We postulate that the interaction of DMPC with DS-5000 in the plane of FF is mediated by Ca²⁺ bridges and results in dehydration of the DMPC polar heads. The interaction between DMPC and DS-5000 in monolayers resulted in slower adsorption and spreading of DMPC molecules at the interface, lower monolayer surface pressure, and penetration of DS-5000 molecules to DMPC monolayers when surface lipid density was higher than 50 Ų per DMPC molecule. The applicability of the FF model for studying the interactions of phospholipids with polysaccharides at interfaces surrounded by bulk solution, and for modeling such interactions in biological systems, e.g. LDL adhesion to the arterial walls, aggregation and fusion of liposomes, etc., is discussed.     

Microscopic visualization of insect cell-bubble interactions. I: Rising bubbles, air-medium interface, and the foam layer.

Through the use of microscopic, high-speed video technology, the interactions of two suspended insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), with air and oxygen bubbles were studied. Events such as cell-bubble attachment, cell-bubble collision, cell transport into the foam layer, and trapping of cells in the foam layer are presented and discussed. Based on these observations and those in a companion paper (Chalmers, J. J.; Bavarian, F. Biotechnol. Prog. 1991, following paper in this issue) and the experimental and theoretical work of other researchers, several mechanisms of cell damage as a result of sparging are presented.     

中药党参类对动物耐疲劳及抗缺氧作用的影响

作者对14个不同种类、不同产地的党参样品水煎液的抗疲劳及耐缺氧作用进行了试验研究,并与党参粗多糖、人参水煎液、人参皂甙进行了比较。党参类具有耐疲劳作用,可增加动物负重游泳时间,党参水煎液与人参皂甙无明显差异,有的甚至优于人参皂甙。党参水煎液腹腔注射可使缺氧小鼠存活时间显著延长,与人参相比无明显差异。     

Treatment with oxandrolone and the durability of effects in older men.

We investigated the effects of the anabolic androgen, oxandrolone, on lean body mass (LBM), muscle size, fat, and maximum voluntary muscle strength, and we determined the durability of effects after treatment was stopped. Thirty-two healthy 60- to 87-yr-old men were randomized to receive 20 mg oxandrolone/day (n = 20) or placebo (n = 12) for 12 wk. Body composition [dual-energy X-ray absorptiometry (DEXA), magnetic resonance imaging, and (2)H(2)O dilution] and muscle strength [1 repetition maximum (1 RM)] were evaluated at baseline and after 12 wk of treatment; body composition (DEXA) and 1-RM strength were then assessed 12 wk after treatment was discontinued (week 24). At week 12, oxandrolone increased LBM by 3.0 +/- 1.5 kg (P < 0.001), total body water by 2.9 +/- 3.7 kg (P = 0.002), and proximal thigh muscle area by 12.4 +/- 8.4 cm(2) (P < 0.001); these increases were greater (P < 0.003) than in the placebo group. Oxandrolone increased 1-RM strength for leg press by 6.7 +/- 6.4% (P < 0.001), leg flexion by 7.0 +/- 7.8% (P < 0.001), chest press by 9.3 +/- 6.7% (P < 0.001), and latissimus pull-down exercises by 5.1 +/- 9.1% (P = 0.02); these increases were greater than placebo. Oxandrolone reduced total (-1.9 +/- 1.0 kg) and trunk fat (-1.3 +/- 0.6 kg; P < 0.001), and these decreases were greater (P < 0.001) than placebo. Twelve weeks after oxandrolone was discontinued (week 24), the increments in LBM and muscle strength were no longer different from baseline (P > 0.15). However, the decreases in total and trunk fat were sustained (-1.5 +/- 1.8, P = 0.001 and -1.0 +/- 1.1 kg, P < 0.001, respectively). Thus oxandrolone induced short-term improvements in LBM, muscle area, and strength, while reducing whole body and trunk adiposity. Anabolic improvements were lost 12 wk after discontinuing oxandrolone, whereas improvements in fat mass were largely sustained.     

EVA regulates thymic stromal organisation and early thymocyte development

Epithelial V-like antigen (EVA) is an immunoglobulin-like adhesion molecule identified in a screen for molecules developmentally regulated at the DN to DP progression in thymocyte development. We show that EVA is expressed during the early stages of thymus organogenesis in both fetal thymic epithelia and T cell precursors, and is progressively downregulated from day 16.5 of embryonic development. In the postnatal thymus, EVA expression is restricted to epithelial cells and is distributed throughout both cortical and medullary thymic regions. Transgenic overexpression of EVA in the thymus cortex resulted in a modified stromal environment, which elicited an increase in organ size and absolute cell number. Although peripheral T lymphocyte numbers are augmented throughout life, no imbalance either in the repertoire, or in the different T cell subsets was detected. Collectively, these data suggest a role for EVA in structural organisation of the thymus and early lymphocyte development.     

Proteomic analysis of the triglyceride-rich lipoprotein-laden foam cells

In hypertriglyceridaemic individuals, atherosclerogenesis is associated with the increased concentrations of very low density lipoprotein (VLDL) and VLDL-associated remnant particles. In vitro studies have suggested that VLDL induces foam cells formation. To reveal the changes of the proteins expression in the process of foam cells formation induced by VLDL, we performed a proteomic analysis of the foam cells based on the stimulation of differentiated THP-1 cells with VLDL. Using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, 14 differentially expressed proteins, containing 8 up-regulated proteins and 6 down-regulated proteins were identified. The proteins are involved in energy metabolism, oxidative stress, cell growth, differentiation and apoptosis, such as adipose differentiation-related protein (ADRP), enolase, S100A11, heat shock protein 27 and so on. In addition, the expression of some selected proteins was confirmed by Western blot and RT-PCR analysis. The results suggest that VLDL not only induces lipid accumulation, but also brings about foam cells diverse characteristics by altering the expression of various proteins.     

The durability of public goods changes the dynamics and nature of social dilemmas

An implicit assumption underpins basic models of the evolution of cooperation, mutualism and altruism: The benefits (or pay-offs) of cooperation and defection are defined by the current frequency or distribution of cooperators. In social dilemmas involving durable public goods (group resources that can persist in the environment-ubiquitous from microbes to humans) this assumption is violated. Here, we examine the consequences of relaxing this assumption, allowing pay-offs to depend on both current and past numbers of cooperators. We explicitly trace the dynamic of a public good created by cooperators, and define pay-offs in terms of the current public good. By raising the importance of cooperative history in determining the current fate of cooperators, durable public goods cause novel dynamics (e.g., transient increases in cooperation in Prisoner's Dilemmas, oscillations in Snowdrift Games, or shifts in invasion thresholds in Stag-hunt Games), while changes in durability can transform one game into another, by moving invasion thresholds for cooperation or conditions for coexistence with defectors. This enlarged view challenges our understanding of social cheats. For instance, groups of cooperators can do worse than groups of defectors, if they inherit fewer public goods, while a rise in defectors no longer entails a loss of social benefits, at least not in the present moment (as highlighted by concerns over environmental lags). Wherever durable public goods have yet to reach a steady state (for instance due to external perturbations), the history of cooperation will define the ongoing dynamics of cooperators.     

EVA: Evaluation of protein structure prediction servers

EVA (http://cubic.bioc.columbia.edu/eva/) is a web server for evaluation of the accuracy of automated protein structure prediction methods. The evaluation is updated automatically each week, to cope with the large number of existing prediction servers and the constant changes in the prediction methods. EVA currently assesses servers for secondary structure prediction, contact prediction, comparative protein structure modelling and threading/fold recognition. Every day, sequences of newly available protein structures in the Protein Data Bank (PDB) are sent to the servers and their predictions are collected. The predictions are then compared to the experimental structures once a week; the results are published on the EVA web pages. Over time, EVA has accumulated prediction results for a large number of proteins, ranging from hundreds to thousands, depending on the prediction method. This large sample assures that methods are compared reliably. As a result, EVA provides useful information to developers as well as users of prediction methods.     

Application of antifungal CFB to increase the durability of cement mortar

Antifungal cement mortar or microbiological calcium carbonate precipitation on cement surface has been investigated as functional concrete research. However, these research concepts have never been fused with each other. In this study, we introduced the antifungal calciteforming bacteria (CFB) Bacillus aryabhattai KNUC205, isolated from an urban tunnel (Daegu, South Korea). The major fungal deteriogens in urban tunnel, Cladosporium sphaerospermum KNUC253, was used as a sensitive fungal strain. B. aryabhattai KNUC205 showed CaCO3 precipitation on B4 medium. Cracked cement mortar pastes were made and neutralized by modified methods. Subsequently, the mixture of B. aryabhattai KNUC205, conidiospore of C. sphaerospermum KNUC253, and B4 agar was applied to cement cracks and incubated at 18 degrees C for 16 days. B. aryabhattai KNUC205 showed fungal growth inhibition against C. sphaerospermum. Furthermore, B. aryabhattai KNUC205 showed crack remediation ability and water permeability reduction of cement mortar pastes. Taken together, these results suggest that the CaCO3 precipitation and antifungal properties of B. aryabhattai KNUC205 could be used as an effective sealing or coating material that can also prevent deteriorative fungal growth. This study is the first application and evaluation research that incorporates calcite formation with antifungal capabilities of microorganisms for an environment-friendly and more effective protection of cement materials. In this research, the conception of microbial construction materials was expanded.     

Wood durability of home-garden teak against brown-rot and white-rot fungi

Natural decay resistance of teak wood grown in home-garden forestry and the factors influencing decay resistance were determined in comparison with that of a typical forest plantation. Accelerated laboratory tests were conducted on 1800 wood samples drawn from 15 trees of three planted sites. Analysis of variance based on a univariate mixed model showed that planted site, fungal species, and their interaction terms were important sources of variation in decay resistance. With increasing decay resistance from centre to periphery of the heartwood, radial position was a critical factor and the interaction effect of fungal species × radial position was significant in influencing the durability. No significant differences were found in decay resistance either between the opposite radii or due to the various possible interaction terms of radii with the site, fungal species and radial position. There were significant differences in decay resistance against brown-rot fungi between wet and dry sites of home-garden teak although differences against white-rot fungi were non-significant among the three planted sites. Polyporus palustris was the more aggressive brown-rot fungus than Gloeophyllum trabeum. The higher susceptibility of wet site home-garden teak to brown-rot decay was associated with a paler colour of the wood and lower extractive content.     

Cell-microcarrier adhesion to gas-liquid interfaces and foam

The interaction of microcarriers, both with and without cells attached, with gas bubbles was studied. These studies consisted of qualitative microscopic observations of microcarriers with bubbles, quantitative measurements of microcarrier entrapment in foam, and quantitative measurements of the effect of bubble rupture at gas-medium interfaces. Ten different "protective additives" were evaluated for their ability to change the dynamic surface tension of the culture media and to prevent microcarrier adhesion to air bubbles during gas sparging and to prevent entrapment in the foam layer. These studies indicate that microcarriers, with and without cells, readily attach to gas-medium interfaces; yet unlike suspended cells, cells attached to microcarriers are not damaged by bubble ruptures at gas-medium interfaces. Only one surfactant was found to substantially prevent microcarrier entrapment in the foam layer; however, this surfactant was toxic to cells. No correlation was observed between surface tension and the prevention of microcarrier adhesion to gas-liquid interfaces. It is suggested that cell damage as a result of sparging in microcarrier cultures is the result of cells, attached to microcarriers, attaching to rising bubbles and then detaching from the microcarrier as this combination rises through the medium. It is further suggested that the hydrodynamic drag force of the rising microcarrier is sufficiently high to remove the bubble-attached cell from the microcarrier.     

The effect of pH on the foam fractionation of beta-glucosidase and cellulase

The surface tension-pH profile of beta-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. This data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic beta-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension-pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension-pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around 10.5, while for cellulase it was between 6 and 9.     

Pi-pi interactions: the geometry and energetics of phenylalanine-phenylalanine interactions in proteins

The geometries of aromatic-aromatic interactions between phenylalanine residues in proteins are analysed in detail and correlated with energy calculations. A new definition of the interplanar angle is important for distinguishing favourable edge-to-face and unfavourable face-to-face orientations. The experimental observations are scattered over a wide range of conformational space, with no strongly preferred single orientation. However, Phe-Phe interactions occur almost exclusively in electrostatically attractive geometries: electrostatically unfavourable regions are only sparsely populated. Electrostatics dominate the geometry of interaction, while van der Waals' interactions are less significant, probably due to the hydrophobic environment of the protein core. The observations on proteins support the Hunter-Sanders rules for pi-pi interactions. In particular, offset stacked geometries, which theory predicts to be favourable, are observed experimentally. For monocyclic aromatics, use of a C-H dipole, the approach used in molecular mechanics calculations, accounts well for these aromatic-aromatic interactions. Comparison with the results obtained from the small molecules database indicates that the protein and small molecule crystal environments are very different.