Membrane damage of liposomes by the mushroom toxin phallolysin

We have investigated the membrane-damaging effect of phallolysin on liposomes varying in phospholipid composition, net charge and physical constitution. Liposomes were prepared from lipids extracted from bovine or human erythrocyte ghosts. The liposomes composed of bovine lipids (the intact cell showing little sensitivity to phallolysin) were found comparably sensitive to those prepared from lipids of human red cells (these cells being of high sensitivity). In addition, artificial mixtures of lipids were used for the preparation of liposomes, consisting of (a) negatively charged phospholipids such as dicetyl phosphate or phosphatidylserine, (b) cholesterol, and (c) either sphingomyelin (as the major component of erythrocytes from ruminants) or phosphatidylcholine (as the major component of erythrocytes from non-ruminants). Again, we found only little difference in the susceptibilities of sphingomyelin- and phosphatidylcholine-containing liposomes. On the other hand, the susceptibility depended on the presence of phospholipids with negative net charges. Omittance of phosphatidylcholine or dicetyl phosphate, or replacement by the positively charged stearylamine, decreased the susceptibility by a factor of more than 20. Finally, we prepared liposomes from dicetyl phosphate, cholesterol and phosphatidylcholine in two physical states: large unilamellar and smaller multilamellar liposomes. The unilamellar liposomes were about 10-times more sensitive to phallolysin. We conclude: (1) Phallolysin damages phospholipid-membranes in the absence of receptor proteins, but high concentrations of the toxin are required. (2) Membrane damage takes place with liposomes containing phosphatidylcholine as well as those containing sphingomyelin. (3) Phallolysin damages only liposomes containing phospholipids with a negative net charge.     

Cytotoxic effects of phallolysin on a human heteroploid cell line

The effect of Phallolysin on cellular growth, macromolecular biosyntheses and cellular membrane structure was analysed using cells from the EUE line. Concentrations of the toxin that do not affect cellular growth, as determined by plating efficiency, have no effect on RNA or protein synthesis, but stimulate DNA synthesis. The doses of Phallolysin that inhibit cell survival do not affect macromolecular biosyntheses, but greatly increase the percentage of cells stainable with Trypan blue after 1 hour of incubation. At the same dose of the toxin the cells, analysed by electron microscopy, show increased vacuolization indicating an alteration of the membrane apparatus.     

Physical and topological properties of circular DNA

Several types of circular DNA molecules are now known. These are classified as single-stranded rings, covalently closed duplex rings, and weakly bonded duplex rings containing an interruption in one or both strands. Single rings are exemplified by the viral DNA from φX174 bacteriophage. Duplex rings appear to exist in a twisted configuration in neutral salt solutions at room temperature. Examples of such molecules are the DNA''s from the papova group of tumor viruses and certain intracellular forms of φX and λ-DNA. These DNA''s have several common properties which derive from the topological requirement that the winding number in such molecules is invariant. They sediment abnormally rapidly in alkaline (denaturing) solvents because of the topological barrier to unwinding. For the same basic reason these DNA''s are thermodynamically more stable than the strand separable DNA''s in thermal and alkaline melting experiments. The introduction of one single strand scission has a profound effect on the properties of closed circular duplex DNA''s. In neutral solutions a scission appears to generate a swivel in the complementary strand at a site in the helix opposite to the scission. The twists are then released and a slower sedimenting, weakly closed circular duplex is formed. Such circular duplexes exhibit normal melting behavior, and in alkali dissociate to form circular and linear single strands which sediment at different velocities. Weakly closed circular duplexes containing an interruption in each strand are formed by intramolecular cyclization of viral λ-DNA. A third kind of weakly closed circular duplex is formed by reannealing single strands derived from circularly permuted T2 DNA. These reconstituted duplexes again contain an interruption in each strand though not necessarily regularly spaced with respect to each other.     

Physical properties of flowing blood

The changes of viscosity, optical reflection and electrical resistivity of blood due to flow are dependent on the orientation and deformation of red cells. From electrical point of view, it can be assumed that blood is suspension of small insulating particles (red cells) in conductive fluid (plasma) when the frequency of supplied voltage is lower than several hundreds KHz. When blood flows, red cells deform and orient in flow direction. Therefore, flowing blood shows anisotropic electrical and optical properties. In steady flow, blood resistivity longitudinal to flow decrease with flow rate, and transverse one increases. Blood flow in living body is not steady but pulsatile. We measured both longitudinal and transverse resistivity changes, optical reflection change and viscosity change of sinusoidally flowing blood in a rectangular conduit. The results are 1) during one period of sinusoidal flow the longitudinal resistivity change is opposite to that of transverse one, 2) the waveform of reflection light change is similar to that of resistance change, and 3) minimum points of both longitudinal resistivity and viscosity changes do not appear at the moment when flow is zero but are delayed. When the amplitude of sinusoidal flow is small and oscillation frequency is high, the phase difference between the zero crossing period of flow and the period of minimum change in resistivity, increases up to 90 degrees. Viscosity of blood decreases with increase of amplitude and frequency of sinusoidal flow.     

Physical properties of microbial suspensions

Physical properties of suspensions of Saccharomyces cerevisiae, Candida utilis and Escherichia coli (density, viscosity and surface tension) were measured in synthetic suspensions formed of centrifuged biomass and supernatants from various stages of batch cultivation in the range from 0 to 10% w/v for yeasts and from 0 to 0.25% w/v for bacteria. Surface tension was also measured in native suspensions in the range of 0 less than or equal to Cm less than or equal to 2.0% w/v. All single cell suspensions were found to be Newtonian in behaviour. Densities strictly obey the mixing law, viscosities of Saccharomyces cerevisiae suspensions were correlated by an empirical relation in dependence of Cm and t, surface tensions were correlated graphically for suspensions of Saccharomyces cerevisiae and Escherichia coli, since experiments with both microorganisms have shown that the previously published approximate correlation can safely be used.     

Physical properties of cholesteryl esters

Cholesteryl esters, the intracellular storage form and intravascular transport form of cholesterol, can exist in crystal, liquid crystal and liquid states. The physical state of cholesteryl esters at physiologic temperatures may be a determinant of their pathogenicity. This review has surveyed saturated aliphatic cholesteryl esters of chain length 1 to 24 carbons and a series of medium-chained unsaturated cholesteryl esters from chain lengths 14 to 24 carbons. A systematic study of transition temperatures by polarizing microscopy and enthalpies by differential scanning calorimetry has provided unifying concepts concerning the phase behavior as a function of chain length and unsaturation. Neat cholesteryl esters show chain-length dependence of transition temperature and enthalpy of both the crystal and liquid crystal transitions. Double bond position along the fatty acyl chain affected stability of the liquid crystal phases; a smectic phase was not observed for any cholesteryl ester with a double bond more proximal than delta 9. 13C NMR spectroscopy in the isotropic liquid phase has provided evidence suggesting a balance of ring-ring vs. chain-chain interactions as a determinant for isotropic liquid----cholesteric vs. isotropic liquid----smectic transitions. Specifically, anisotropic molecular motions of the steroid ring are greater for cholesteryl esters forming a cholesteric phase than a smectic phase from the melt. Chain-chain interactions apparently predominate in smectic phase formation. The X-ray diffraction patterns of cholesteryl esters as a function of chain length reveal several isostructural series and known single crystal data are presented. A chain length depending on the periodicity of the smectic phase is observed which may be different for saturated vs. unsaturated esters. In summary, the phase behavior of cholesteryl ester molecules is complex and cannot be determined a priori from the phase behavior of component cholesterol and fatty acid. The data presented here should provide insight into the biological behavior of this lipid class.     

Physical properties of phage 299

Summary Phage 299, on equilibrium sedimentation in CsCl, gives 3 major bands whose relative proportions depend on growth conditions. One band is whole heads without tails, the other two are infectious phage of differing degrees of disarray and stability. Electron micrographs show that the infectious phage has a head about 60 nm across, probably icosahedral in shape, and a straight tail of approximately 140 nm in length. The tail assemblies appear defective and incomplete. Sedimentation in a sucrose gradient of the DNA extracted from phage 299 is monodisperse with a molecular weight of 20.6±1.5×10⁶ daltons based on comparison with λ and 186 phage DNA’s. The DNA has a base composition of 51.7% guanine and cytosine as determined by bouyant density in CsCl. A comparison of its denaturation behavior by analysis of the hyperchromic shift at 260 nm with that of phage P2 suggests a considerable number of common characteristics, and an absence of a low guanine and cytosine portion on the part of 299 which amounts to approximately 10% of the total DNA.     

Physical properties of polyethyleneimine-alginate gels

Summary The procedure for preparation of polyethyleneimine (PEI)-alginate gel and some of its physical properties are presented. The rigidity modulus (G) of the gel appears to increase to the second power of PEI concentration. Shrinkage is linearly related to pH up to pH 6.0. G increases as the pH is lowered, and decreases after freezing. The gels does not dissolve on heating and are strong, but fairly brittle. They are resistant to proteolytic enzymes.     

Physical properties of fungal chitosan

Fungi are promising alternative sources of chitosan. This study evaluated the physical properties of fungal chitosan from Absidia coerulea (AF 93105), Mucor rouxii (Ag 92033), and Rhizopus oryzae (Ag 92033). FT-IR and X-ray diffraction of the extracted products showed typical chitosan peak distributions which confirmed the extracted products to be chitosan. All of their glucosamine contents and degrees of deacetylation (DD) were over 80%, not showing obvious differences respectively. However, differences had been observed in their molecular weight (M_w), ranging from 6.6  to 560 kDa. The results of this study demonstrated that different fungi could produce different M_w chitosan with high DD and high purity.     

Physical properties of microbial suspensions

A way of characterizing cell size distribution in suspensions of single-cell microorganisms is suggested, based on the first and second moments around origin. Suspension density may be predicted on the basis of the mixing law and the density of microbial dry matter, for suspensions ofSaccharomyces cerevisiae, Candida lipolytica, andAspergillus niger. Newtonian viscosities were correlated using regression analysis by η=Aexp(BC _m)exp(D/t); another correlation is presented for the calculation of surface tension in single-cell microbial suspensions. All the relations are valid in the range of concentrations up to 15% (w/v) and for temperatures between 15° and 35°C. The formulae presented may be used in other hydraulic studies.     

Physical and biological properties of homophilic therapeutic antibodies

Homophilic antibodies have been discovered in mice and primates and can also be engineered. Compared to conventional antibodies, homophilic antibodies form lattices on targets leading to enhanced binding via polyvalent attachment. Previously, we have observed a paradoxical dose/potency effect with an engineered homophilic antibody against a human lung cancer tumor. Here, we have investigated some biophysical properties of homophilic antibodies and also studied the inhibition of human tumor growth in a xenograft model using homophilic Herceptin. Dimerization and viscosity of two homophilic antibodies are greater at physiological temperature than at 4°C. Similarly, binding to solid-phase antigen is greater at 37°C than at room temperature or 4°C. Dimer formation is higher at therapeutic concentration, supporting the notion that preformed dimers in solution are the effective molecular species responsible for polyvalent target binding and enhanced therapeutic potency.