Study of binding between protein A and immunoglobulin G using a surface tension probe

Molecular interactions and binding are one of the most important and fundamental properties in the study of biochemical and biomedical systems. The understanding of such interactions and binding among biomolecules forms the basis for the design and processing of many biotechnological applications, such as bioseparation and immunoadsorption. In this study, we present a novel method to probe molecular interactions and binding based on surface tension measurement. This method complements conventional techniques, which are largely based on optical, spectroscopic, fluorescence polarization, chromatographic or atomic force microscopy measurements, by being definite in determining molecular binding ratio and flexible in sample preparation. Both dynamic and equilibrium (or quasi-equilibrium) information on molecular binding can be obtained through dynamic and equilibrium surface tension measurements. For an important pair of biological ligand and ligate, Protein A and immunoglobulin G (IgG), the existence of molecular interactions and the binding ratio of 1:2 have been determined unequivocally with the proposed surface tension method. These results are confirmed/supported by a mass balance calculation and spectrophotometry experiment. In addition, adsorption isotherms for Protein A and IgG separately at the air/water interface have been established with the dynamic surface tension measurements. The results show that the Langmuir isotherm equation can describe the adsorption data satisfactorily for both Protein A and IgG solutions.     

STUDIES ON THE FORMATION AND IONIZATION OF THE COMPOUNDS OF CASEIN WITH ALKALI : IV. THE TRANSPORT NUMBERS OF THE COMPOUNDS OF CASEIN WITH THE ALKALI EARTH ELEMENTS.

1. Data from the results of transference experiments on solutions of the alkaline earth caseinates are given. 2. The data support the idea that part of the alkaline earth element is held by the casein in the form of complex ions. 3. Grounds are given for believing that the complex anions have a definite composition.     

Survismeter--type I and II for surface tension, viscosity measurements of liquids for academic, and research and development studies

For centuries surface tension (gamma) and viscosity (eta) data have been measured with individual instruments consuming much time and materials. Thus the two different types of survismeters have been designed and made of borosil glass material for surface tension and viscosity data to rationalize frictional and cohesive forces, respectively. Friccohesity (sigma (sm(-1), second per meter)) is derived from Fric of frictional and cohesity of cohesive forces of the liquid respectively, and denoted by rational coefficient a eta/gamma (sigma). The values of the friccohesity are correlated to the dipole moment of liquids and their plot gives a standard calibration curve along with an equation of the curve with definite values of the coefficients, the friccohesity values are put on the calibration curve to retrieve the dipole moment values. Here, the sigma values for each of dimethylformamide, dimethylsulfoxide and acetronitrile solvents along with their 0.05, 0.10 and 0.20 mol kg(-1) aqueous solutions, respectively, were determined at 293.15K and plotted against their dipole moment values for standard calibration curve. The range of the dipole moment values for calibration curve is pre-decided and the sigma values for homogenous solutions of compositions below their saturation point can be measured with +/-1 x 10(-5)sm(-1).     

Freezing of xylem sap without cavitation

Freezing of stem sections and entire twigs of hemlock (Tsuga canadensis) has been demonstrated to occur without increasing the resistance to the movement of water through the frozen part after rewarming. This was interpreted to mean that freezing did not produce cavitation in the xylem sap even though A) the sap was unquestionably frozen; B) it contained dissolved gases; and C) it was under tension before freezing and after. Freezing stem sections of some other evergreen gymnosperms during the summer again produced no evidence for cavitation of the xylem sap. On the other hand, freezing stem sections of some angiosperms invariably increased the resistance to sap flow leading to wilting and death in a few hours when the sap tension was at normal daytime values at the time of freezing. These results were interpreted to mean that the bordered pits on the tracheids of gymnosperms function to isolate the freezing sap in each tracheid so that the expansion of water upon freezing not only eliminates any existing tension but also develops positive pressure in the sap. Dissolved gases frozen out of solution may then be redissolved under this positive pressure as melting occurs. As the bubbles are reduced in size by this ice pressure developed in an isolated tracheid, further pressure is applied by the surface tension of the water against air. If the bubbles are redissolved or are reduced to sufficient small size by the time the tension returns to the sap as the last ice crystals melt, then the internal pressure from surface tension in any existing small bubbles may exceed the hydrostatic tension of the melted sap and the bubbles cannot expand and will continue to dissolve.     

Patterns of occurrence of corticolous myxomycetes on white oak trees of two different size classes

Data were obtained on the assemblages of corticolous myxomycetes (plasmodial slime moulds or myxogastrids) associated with the bark surface of living white oak (Quercus alba) trees from two different size classes. Bark samples obtained from larger trees were characterized by higher values for both species richness and diversity when compared to those collected from smaller trees. This might have been expected since the former possess a larger surface area and presumably have persisted over a longer period of time. However, the myxomycete assemblage associated with smaller trees was appreciably different and did not simply represent a depauperate version of the assemblage associated with larger trees. This suggests that the differences observed between size classes cannot be simply attributed to size alone.     

Fish toxicity and surface tension of non-ionic surfactants: investigations of antifoam agents

Fish toxicity of etho-nonylphenol and of two non-ionic antifoam agents was tested using rainbow trout, Sulmo guirdneri , in 24 h tests. The surface tension of these three compounds was found to depend on concentration and ageing time of the test solutions. The toxicity of the surfactant was about twice as high as known values, with a LC⁵⁰ of 8.5 mg/1 at 15°C. The addition of antifoam agents to the surfactant solutions further lowered the surface tension but did not alter substantially the fish toxicity of the surfactant. Contrary to expectation, the fish survived in concentrated antifoam solutions at surface tensions as low as 31 dyn/cm for more than 24 h without any apparent damage. The relevance of the surface tension regarding the fish toxicity of detergents in general is critically discussed.     

An analysis of the shapes of a cell during division with particular reference to the role of surface tension

An expression is given for the contribution to the relative elongation by any volume element or its surface. The case in which only a constant surface tension is acting is considered in greater detail. From measurements of an egg in several stages, the empirical variation of an expression proportional to the above function is obtained. In the constricting region and near the ends of the cells, the change of shape is opposite to that which would be due to a constant surface tension alone. The effect of streaming which may arise from a variable surface tension is considered. The effect of forces which arise in this manner may be sufficient to explain the discrepancy in the constricting region if the streaming is considered due largely to such a variable surface tension.     

Nucleoli in the cells of the erythrocyte and granulocyte series in the albino rat

In albino rats, as in other mammals, nucleoli with nucleolonemata and compact nucleoli in the stem cells of the erythrocyte and granulocyte series are progressively replaced during maturation and differentiation first by ring shaped nucleoli and then by micronucleoli with low and finally with inhibited RNA synthesis. There are, however, differences between the nucleolar coefficient values and the proportion of ring shaped nucleoli and micronucleoli in maturing neutrophils in the albino rat, the mouse and man. In the case of the erythroblasts, the differences between the proportion of various nucleolar types in the three given species are smaller. The results thus indicate that the developmental trend of nucleolar changes related to cell maturation and differentiation is the same, but that there are interspecies differences because of which the results obtained in one species cannot be applied mechanically to another, i.e. results obtained in a laboratory animal cannot be applied automatically to man.     

THE NATURE OF THE GROWTH RATE

1. The growth rate of organisms may be considered as a chemical reaction which gives the mature organism as its end-product. The organism grows at a definite rate which is, at any moment, proportional to the amount of growth yet to be made. 2. Shoots of young pear trees measured at weekly intervals during the growing season showed a rate similar to that of an autocatalytic reaction. 3. Young walnut trees showed distinct cycles of growth in a single season, but the growth in each cycle proceeded at a rate corresponding to an autocatalytic reaction. 4. The growth rate follows a definite, quantitative course though judged by different criteria. Data are presented for maize in which green weight, dry weight, and height of the plant are used. Data for cattle show that either weight or height of the animal may be used as a criterion.     

Axisymmetric Drop Shape Analysis for Estimating the Surface Tension of Cell Aggregates by Centrifugation

Biological tissues behave in certain respects like liquids. Consequently, the surface tension concept can be used to explain aspects of the in vitro and in vivo behavior of multicellular aggregates. Unfortunately, conventional methods of surface tension measurement cannot be readily applied to small cell aggregates. This difficulty can be overcome by an experimentally straightforward method consisting of centrifugation followed by axisymmetric drop shape analysis (ADSA). Since the aggregates typically show roughness, standard ADSA cannot be applied and we introduce a novel numerical method called ADSA-IP (ADSA for imperfect profile) for this purpose. To examine the new methodology, embryonic tissues from the gastrula of the frog, Xenopus laevis, deformed in the centrifuge are used. It is confirmed that surface tension measurements are independent of centrifugal force and aggregate size. Surface tension is measured for ectodermal cells in four sample batches, and varies between 1.1 and 7.7 mJ/m². Surface tension is also measured for aggregates of cells expressing cytoplasmically truncated EP/C-cadherin, and is approximately half as large. In parallel, such aggregates show a reduction in convergent extension-driven elongation after activin treatment, reflecting diminished intercellular cohesion.     

Polyelectrolyte/amphiphile interaction studied by surface tension measurements

The interaction of κ-carrageenan with three positively charged drug molecules with amphiphile character has been examined using surface tension measurements. The surface tension was measured by the pendant drop method which makes possible the determination at an apparent steady state which is important for polymeric systems. The results are compared with adsorption isotherms from dialysis equilibrium. The surface tension data, show that the presence of κ-carrageenan in the amphiphile solutions leads to an increased and pronounced lowering of the surface tension in a low concentration range of amphiphile. It is also shown that not only the hydrophobicity of the amphiphile but also the structure of the polyelectrolyte (charge density and helix-coil structure) largely determine the extent of interaction.     

Estimation of muscle active state

A method is presented for the estimation of the complete time course of muscle active state. The method is based on the selection of a proper model for the muscle, consisting of linear and non-linear components, and on the estimation of its parameters from a simple experiment. The model's parameters are estimated, using the least square method, from measurements of a tetanized muscle's response to a change of its length. The time course of the active state is calculated from an isometric twitch tension response of the same muscle. The twitch tension response is taken as the system's output, and the active state as its input. The latter can be estimated since the system parameters have already been estimated from the tetanized muscle experiment. Experiments were performed on the gastrocnemius muscle of frogs and cats. Results are given for the whole active state time course of these muscles. The results show that the peak active state force does not reach tetanic value, and a negative force is generated during the relaxation period. Additional experiments were carried out with the purpose of verifying the existence of this force; however, no conclusive results were obtained.This research was supported by the Julius Silver Institute of Bio-Medical Engineering Sciences, Grant 050-304     

The Marchi Method

An investigation has been made of the poor penetration obtained by the Marchi method. Use of surface tension depressants or perfusion technics showed no marked improvement. Optimum results cannot be achieved with slices of tissue above 3 mm. in thickness. Another cause of faulty staining is the failure to maintain an adequate strength of OsO₄ in the Marchi mixture. The concentration of this chemical should never be allowed to drop below 0.25%. A chemical method for determining the amount of OsO₄ present in the staining mixture and based upon the Alvarez test is described. This method can also be utilized to reclaim partially exhausted staining solutions. Remarks are included upon the KIO₃ method of Busch.     

TENSION AT THE HIGHLY STRETCHED SURFACE OF SEA-URCHIN EGGS*

Unfertilized eggs of the sea urchin, Paracentrotus lividus, were placed between two parallel plates and flattened by a definite force to 20% of their original diameter, with two-fold increase in their surface area. The resulting tension at their surface was calculated from the relation of force and deformation. In spite of this extensive stretching, the tension was found to be not more than 0.2 dyne/cm, while under conditions involving mild stretching (3%) the tension still amounted to 0.12 dyne/cm. These results do not support Mela's theory (7, 8), which predicts a transition of the mechanical properties of the egg surface from a ‘subelastic’ to ‘elastic’ state when the surface is stretched to beyond 34% of its initial area.     

Comparison of methods to detect biosurfactant production by diverse microorganisms

Three methods to detect biosurfactant production, drop collapse, oil spreading, and blood agar lysis, were compared for their ease of use and reliability in relation to the ability of the cultures to reduce surface tension. The three methods were used to test for biosurfactant production in 205 environmental strains with different phylogenetic affiliations. Surface tension of select strains that gave conflicting results with the above three methods was also measured. Sixteen percent of the strains that lysed blood agar tested negative for biosurfactant production with the other two methods and had little reduction in surface tension (values above 60 mN/m). Thirty eight percent of the strains that did not lyse blood agar tested positive for biosurfactant production with the other two methods and had surface tension values as low as 35 mN/m. There was a very strong, negative, linear correlation between the diameter of clear zone obtained with the oil spreading technique and surface tension (rs = -0.959) and a weaker negative correlation between drop collapse method and surface tension (rs = -0.82), suggesting that the oil spreading technique better predicted biosurfactant production than the drop collapse method. The use of the drop collapse method as a primary method to detect biosurfactant producers, followed by the determination of the biosurfactant concentration using the oil spreading technique, constitutes a quick and easy protocol to screen and quantify biosurfactant production. The large number of false negatives and positives obtained with the blood agar lysis method and its poor correlation to surface tension (rs = -0.15) demonstrated that it is not a reliable method to detect biosurfactant production.     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.     

PHYLOGENETIC ANALYSES OF THE CORRELATED EVOLUTION OF CONTINUOUS CHARACTERS: A SIMULATION STUDY

We use computer simulation to compare the statistical properties of several methods that have been proposed for estimating the evolutionary correlation between two continuous traits, and define alternative evolutionary correlations that may be of interest. We focus on Felsenstein's (1985) method and some variations of it and on several “minimum evolution” methods (of which the procedure of Huey and Bennett [1987] is a special case), as compared with a nonphylogenetic correlation. The last, a simple correlation of trait values across the tips of a phylogeny, virtually always yields inflated Type I error rates, relatively low power, and relatively poor estimates of evolutionary correlations. We therefore cannot recommend its use. In contrast, Felsenstein's (1985) method yields acceptable significance tests, high power, and good estimates of what we term the input correlation and the standardized realized evolutionary correlation, given complete phylogenetic information and knowledge of the rate and mode of character change (e.g., gradual and proportional to time [“Brownian motion”] or punctuational, with change only at speciation events). Inaccurate branch length information may affect any method adversely, but only rarely does it cause Felsenstein's (1985) method to perform worse than do the others tested. Other proposed methods generally yield inflated Type I error rates and have lower power. However, certain minimum evolution methods (although not the specific procedure used by Huey and Bennett [1987]) often provide more accurate estimates of what we term the unstandardized realized evolutionary correlation, and their use is recommended when estimation of this correlation is desired. We also demonstrate how correct Type I error rates can be obtained for any method by reference to an empirical null distribution derived from computer simulations, and provide practical suggestions on choosing an analytical method, based both on the evolutionary correlation of interest and on the availability of branch lengths and knowledge of the model of evolutionary change appropriate for the characters being analyzed. Computer programs that implement the various methods and that will simulate (correlated) character evolution along a known phylogeny are available from the authors on request. These programs can be used to test the effectiveness of any new methods that might be proposed, and to check the generality of our conclusions with regard to other phylogenies.     

Effects of Storing Initiated 2-Hydroxyethyl Methacrylate Solutions for Embedding Tissues for Light Microscopy: Some Practical Implications

The effects of storing 2-hydroxy-ethyl methacrylate (HEMA) solutions for embedding tissues for light microscopy were studied using three commercially available HEMA embedding kits: Technovit 7100, Technovit 8100, and JB-4. These HEMA solutions were examined at various times of storage over a period of one year using a panel of physicochemical techniques including gas chromatography, tltration, viscosimetry, determination of the maximum polymerization temperature and the time required to reach the maximum temperature, and detection of degradation products of HEMA monomers by histochemical procedures. The quality of the resin blocks was examined by the observation of mini-folds in sections. Data obtained from these tests showed that the release of by-products as a result of the degradation of the HEMA monomer during storage of HEMA solutions does not occur. Development of cross-linking agents by transesterification of HEMA monomer was not detected either. Gradual decrease of the inhibitor concentration during storage proved to be the main cause of the reduction of shelf-life of HEMA solutions. Inconsistent tissue infiltration after storage may be due to decreased rates of tissue penetration as a result of HEMA chain lengthening. Guidelines for safe and economical handling of HEMA mixtures are given.     

Geometry of the five-membered ring mathematical demonstration of the pseudorotation formulae

The geometrical relations between the 15 typical parameters (bond lengths and angles, torsion angles) of a five-membered ring are derived for any ring then for a regular one. It is demonstrated that for the case of the 20 symmetrical C ₂ and C _sconformations, only geometrical considerations are needed to obtain the pseudorotation formulae for the torsion angles. However, the puckering intensity as well as the bond angle values cannot be expressed from geometrical constraints alone but would require energetical considerations.