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Abnormal membrane surface properties during maturation of rat reticulocytes elicited by bleeding as measured by partition in two-polymer aqueous phases.

Partition of cells in two-polymer aqueous phases is an extremely sensitive method for the separation of cells and for tracing subtle changes in the cells' membrane surface properties (primarily surface charge) as a function of in vivo processes (e.g. differentiation, maturation, ageing). Combining isotope labelling and cell countercurrent distribution techniques we have examined the membrane surface properties of rat reticulocytes produced in response to erythroid stress by bleeding. It was found that the rapid increase in the partition coefficient of normal reticulocytes subsequent to release into the peripheral blood (which reflects a rapid increase in membrane surface charge) is absent in reticulocytes produced in response to bleeding. In this way the reticulocytes behave like those produced in response to repeated phenylhydrazine injections. 'Stress reticulocytes' never mature to erythrocytes having normal membrane surface properties as judged by partition. The experiments show that our previous results are not due to the phenylhydrazine per se but rather to the production of 'stress reticulocytes'. Further, if remodelling of the phlebotomy-induced reticulocyte cell membrane occurs in the circulation as has been suggested it does not lead to a cell with normal membrane surface properties. Whether the abnormal membrane surface properties of 'stress reticulocytes' and of the erythrocytes to which they give rise affect the cells' life-span is not clear since phenylhydrazine-induced 'stress reticulocytes' have been reported to have at least a component of short-lived cells while the phlebotomy-induced cells are said to have a virtually normal life-span. It is likely that reticulocytes produced in severe anaemias, in general, mature to erythrocytes having abnormal surface properties (i.e. lower membrane surface charge).

Animals

Surface properties of cells of Pseudomonas aeruginosa possessing R-factor-mediated resistance to gentamicin.

The surface properties of cells of Pseudomonas aeruginosa which are sensitive to gentamicin are markedly different from those which are resistant to gentamicin. Cells of four out of five strains in which gentamicin-resistance is R-factor-mediated have electrokinetic properties characteristic of gentamicin-sensitive cells. The surface properties of transconjugant strains are indistinguishable from those of the original acceptor strain. Cells of the remaining R-factor donor strain (which transferred very low gentamicin-resistance) exhibited surface properties characteristic of gentamicin-resistant cells. Gentamicin-resistance in cells of this strain is thus the result of at least two different mechanisms. The results are discussed in terms of possible alternative mechanisms of resistance.

Carbenicillin

Cell surface properties of L1210 leukaemia cells.

The surface properties of vincristine-colchicine sensitive and resistant L1210 leukaemic cells have been studied using concanavalin A mediated agglutination assay as well as electron microscopic visualization of concanavalin A receptors. 3H-colchicine uptake by the sensitive and resistant lines has also been compared. The resistant L1210 leukaemic cells proved less agglutinable than the sensitive ones at the same concanavalin A concentration. Previous treatments with either colchicine, vincristine or chlorpromazine caused a marked decrease in the agglutinability of the sensitive L1210 leukaemic cells, while agglutination of the resistant ones was lowered slightly by the same treatments. The 3H-colchicine uptake of the sensitive cells was three times higher than that of the resistant ones.

Animals

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties.

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distribution of 14C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.

Animals

Surface properties of the amniotic fluid in normal pregnancy.

The surface properties of the amniotic fluid were determined in 96 normal pregnancies between weeks 23 and 41. The y-max, y-min, hysteresis areaa, and stability index were used as parameters for evaluation of the surface tension are diagrams. These results were plotted against the gestation week and a curve for the normal course of surface tension of the amniotic fluid during the second half of gestation was obtained. The influence of addition of blood, vernix, and meconium to amniotic fluid as well as that of changes in the pH and dilution were examined. The results were unaffected by either blood, vernix, or pH changes. The slight scatter of the y-min values makes this parameter particularly suitable for determining fetal lung maturity.

Amniotic Fluid

Surface properties of amniotic fluid and fetal lung maturity.

Surface activity of 75 amniotic fluid samples from pregnancies which ended before term or at term with small-for-date newborns was measured in the surface balance. The results were correlated with the clinical symptomatology and the birth weight of the newborn. The surface properties of the lungs of those infants who had died were assessed by the buble stability method (Pattle), by recording of pressure-volume diagram and by measurements of surface activity of lung extracts. The results indicate that gamma-min is the essential parameter in determining the degree of fetal lung maturity. In cases with gamma-min values of amniotic fluid above 27 dyn/cm, a 100% respiratory distress syndrome (RDS) incidence is to be expected; values between 23 and 27 dyn/cm have a 70% values between 17 and 23 dyn/cm have a 30% incidence of RDS and in all cases with values below 17 dyn/cm, RDS may be rules out. When induced delivery before term is considered, this method of determining the degree of fetal lung maturity is, therefore, of significant prognostic reliability.

Amniotic Fluid

Changes in structure and hydrophobic surface properties of beta-lactoglobulin determined by partition in aqueous two-phase polymeric systems.

The non-polar surface properties of beta-lactoglobulin and especially its interaction with poly(ethylene glycol)-bound palmitate has been studied as a function of pH, temperature and protein concentration. The maximum interaction between beta-lactoglobulin and polymer-bound palmitate occurs at pH 4.3 and pH 7.8. The change in conformation of beta-lactoglobulin around pH 7.5 seems to involve exposure of apolar amino acids to the solvent which results in an increased affinity for hydrocarbons. This is contrary to the situation at pH 4.8--6.0 where the corresponding change in conformation does not affect the protein-hydrocarbon interaction. The results suggest that partition studies in an aqueous two-phase system is a very useful tool to detect changes in conformation and aggregation and to characterize the corresponding hydrophobic surface properties of a protein.

Animals

Membrane surface properties of sheep erythrocytes, an immunological reagent, after different treatments as reflected by partition in two-polymer aqueous phases.

Sheep erythrocytes (E) which, with or without certain treatments, are currently used as "immunological reagents" to detect cells with specific receptors (by rosette-formation) have been partitioned in two-polymer aqueous-phase systems selected so as to reflect charge-associated or lipid-related membrane surface properties. We have found that the partitioning behavior of E is not affected in these phases by reacting the cells with anti-E antibody (either IgG or IgM), forming EA. The additional binding of complement to the cell-antibody complex, forming EAC, results, however, in a marked decrease in the partition coefficient, K. Apparently both the charge-associated and hydrophobic properties reflected by partitioning remain accessible to the phase polymers when the cells are coated with antibody, but are not with the addition of complement. It is interesting that EA can still rosette with T-lymphocytes (14), a property of E, while the additional coating with complement results in EAC which does not appreciably do so (26). Neuraminidase or trypsin treatments of E, which yield Es having quite different rosetting properties with T-lymphocytes (14), cause increased Ks and unchanged Ks, respectively, in phases reflecting lipid-related surface properties. Either treatment causes reduced Ks of E in charged-phase systems. Neuraminidase treatment also results in a reduced electrophoretic mobility of E, while trypsin treatment is not detectable by cell electrophoresis (25). We are currently studying the possible usefulness of employing cell electrophoresis and cell partitioning in charged-phase systems jointly to obtain information on events occurring at the shear plane versus those occurring deeper in the membrane.

Animals

Surface properties of monomolecular films of oxidized and reduced cytochrome c and f.

The surface properties of monomolecular films of oxidized and reduced cytochromes f and c were measured at an air-water interface. Area/molecular (A) and surface potential (deltaV) for oxidized and reduced forms of the cytochromes were measured as a function of pH. Oxidized cyt f has a maximum for both A and deltaV at pH 7.5. At a surface pressure of 6 dyn/cm the maximum A equals 2600 plus or minus 50 A2 and the maximum deltaV equals 200 plus or minus 10 mV. Reduced cyt f as a function of pH has a minimum value for both A (2200 A2) and deltaV (95 mV). Oxidized cyt c as a function of pH has minima for A (140 A2) and deltaV (188 mV) at pH 7.0 and 7.3, respectively. On the other hand, reduced cyt has maximum values for A (220 A2) and deltaV (260 mV) at pH 7.0 and 7.3, respectively.

Animals

Surface induced in vitro angiogenesis: surface property is a determinant of angiogenesis.

The control of cellular responses on substrate surfaces is essential for logical surface design aiming at endothelialized, vital implant devices. In this paper, the surface property that alters cell adhesion, spreading, migration, and proliferation processes is shown to be a determinant of endothelial cell assembly or angiogenesis in vitro. This was clearly demonstrated on slightly hydrophobic cellulosic surfaces, which induced organized three-dimensional cellular assemblies of bovine thoracic endothelial cells. The results indicated that this was driven by enhanced migratory response and/or retraction or involution of two-dimensional adherent cells, in which cell-cell interaction was enforced in a time dependent fashion. The present study strongly suggests that the mechanism leading to in vitro angiogenesis is primarily due to a weak cell-substrate interaction relative to cell-cell interaction.

Animals

Surface properties and functional characteristics of infiltrating cells harvested from acutely rejecting cardiac allografts in inbred rats.

Viable, functioning effector cells exerting donor specific cytotoxic properties have been removed, with their surface properties intact, from acutely rejecting cardiac allografts in rats to define immunological events occurring within the grafts themselves. Macrophages comprised 15 to 25% of the cells harvested; lymphocytes comprised about 75%. A few polymorphonuclear leukocytes (approximately 5%) were present consistently. Lymphocytes bearing surface Ig, B cells, made up 35 to 47% of the mononuclear cells collected. The remaining lymphocytes were presumptive T cells. The surfact attributes of the infiltrating cells were compared to those of cells from recipient peripheral blood, spleen and lymph nodes, and to those from isografted controls. Many of the B lymphocytes possessed Fc receptors but were virtually unable to form erythrocyteantibody complement (EAC) rosettes, in contrast to splenocytes which formed approximately 30% EAC rosettes. Specific cytotoxicity against donor alloantigen-bearing cells, as tested in a 51Cr release assay, was shown to be a T cell function by serial fractionation experiments. Peak cytotoxicity of infiltrating T cells occurred early during the rejection process, while that of cells from recipient blood and lymphoid tissues occurred only following complete graft destruction. Antibody-dependent lymphocyte-mediated cytotoxicity (Ab-LMC) was demonstrated following gentle trypsinization or overnight incubation of the cells. Differing effector cell populations could be distinguished by treatment of anti-Ig and complement; direct donor-specific lymphocyte-mediated cytotoxicity was not disrupted by this treatment, while Ab-LMC was abolished.

Animals

Influenza A virus interaction with murine lymphocytes. II. Changes in lymphocyte surface properties induced by influenza virus A/Japan 305 (H2N2).

This report describes changes in surface properties of rat thoracic duct lymphocytes (TDL) induced by influenza virus A/Japan 305 (H2N2). After incubation with virus at 37 degrees C, sialic acids were released from the membranes of TDL WITH CONCOMItant reduction in their mean electrophoretic mobility. Autoradiographs of TDL which had been incubated with 125I virus at 4 degrees C showed that the particles attached to nearly all the lymphocytes although there was variation in the amount of virus bound per cell. Attachment occurred rapidly with maximum labeling evident after incubation at 4 degrees C for 5 min. Incubation at 37 degrees C for 1 hr resulted in a 10 to 18% reduction in the incidence of labeled TDL suggesting that some virus eluted spontaneously. When 125I-virus-treated TDL were incubated in syngeneic serum, substantial amounts of virus eluted reducing the incidence of labeled cells by 50%. In addition, evidence was obtained by indirect immunofluorescence that syngeneic serum contains antibodies which react with sites on the lymphocyte surface exposed by the viral neuraminidase.

Animals

Surface properties of bloodstream Trypanosomes (Trypanosoma brucei).

The African trypanosome (Trypanosoma brucei brucei) in its bloodstream phase has been found to possess rather unique surface properties. Results of lectin and iron colloid binding studies have indicated that the parasite's surface coat is devoid of many types of exposed carbohydrate residues which commonly are expressed on a variety of mammalian cell types. These include: glucose, mannose, galactose, n-acetylglucosamine, n-acetylgalactosamine, and neuraminic acid. A number of these carbohydrates (E.G., glucose, mannose, galactose and n-acetylgalactosamine) are, however, buried within the surface coat as evidenced by lectin binding to trypsinized parasites. The surface of well preserved parasites also lack exposed sialic acid and phospholipid moieties. Moribund parasites (or trypsinzed organisms) with altered surface coats were found to possess well defined phospholipid regions, but not negatively charged sialyl residues. Results presented tend to suggest that carbohydrates and phospholipids are not present in the outermost portion of the glycocalyx, thus exerting little or no influence on surface-mediated biological activities. However, the presence of such components on the plasmalemma is presumed to be structurally significant; especially during those developmental phases where the glycocalyx is absent, i.e., fly midgut phase.

Binding Sites

[Relationship between the surface properties of phosphoglycerides and the structure of the phosphorus part of the molecule].

Comparative studies have been made on surface properties of phospholipids with different chemical composition and different structure of the hydrophylic moiety of the molecule. Substitution of phosphoroyl oxygen by sulfur, as in diacetyl lecithins, makes thiolecithin molecules in monolayer more compact. In case of an analogous substitution of oxygen by sulfur in phosphodiglycerides, packing of molecules in a monolayer remains unaffected. It may be suggested that this fact is associated with different position of hydrophobic radicals in the compounds investigated. In contrast to classic neutral and phosphorus-containing phosphoglycerides, diolphospholipids form more condensed monolayers. It was shown that alpha- and beta-phosphatidic acids with the same composition exhibit identical surface activity. Incorporation of hydrophobic groups of aromatic type into synthetic phospholipid analogues increases surface activity, the effect being dependent on the location of these groups with the molecule.

Chemical Phenomena

[Apparatus for studying the surface properties of pulmonary extracts].

The author describes a unit for studying the surface properties of the pulmonary extracts by Wilhelmy's principle; the unit consists of a two-coordinate self-recorder, compensatory amplifier, modified Wilhelmy's balance, a rectifier and a stabilizer. The unit permits automatic recording of the dependence of surface tension of the extract upon the area of its surface.

Chemistry, Physical

Surface properties of mussel adhesive protein component films.

Mussel adhesive protein (MAP) is the adhesive agent used by the blue sea mussel (Mytilus edulis) to attach the animal to various underwater surfaces. It is composed of 75-->85 repeating decameric units with the reported primary sequence NH2-A(1)-K(2)-P(3)-S(4)-Y(5)-Hyp(6)-Hyp(7)-T(8)-DOPA(9)-K(10)-COOH. This study identifies and compares the surface properties of the decameric unit, selected fragments and individual amino acid constituents with the complete MAP preparation. These molecular systems were examined: (a) in the solid state as thin films formed on germanium substrata using multiple-attenuated-internal-reflectance infrared (MAIR-IR) spectroscopy, ellipsometry and contact angle analysis; and (b) in the solution state using circular dichroism (CD) spectroscopy. Extensive molecular modelling of the decamer was performed making integral use of the experimentally derived data. These cumulative semi-empirical and empirical results suggest a conformation for the decamer that closely associates the L-DOPA and tyrosine residues with the solid substratum. This model provides the first representation of MAP derived from a rational integration of theoretical and experimental data. On the basis of this model, a possible explanation for the bioadhesive properties of MAP is suggested.

Amino Acid Sequence

Fractionation of microsomal membranes on the basis of their surface properties.

Partition in dextran-poly(ethylene glycol) aqueous-phase systems can be used for both membrane subfractionation and gaining information on membrane surface properties [H. Walter (1977) in Methods of Cell Separation (Catsimpoolas, N., ed.), vol. 1, pp. 307-354, Plenum, New York]. Smooth, light rough and heavy rough rat liver microsome (obtained by sucrose-density-gradient centrifugation) were subjected to countercurrent distribution in such a system. Smooth microsomal membranes had the highest, heavy rough microsomal membranes the lowest and light rough microsomal membranes an intermediate partition coefficient. The separation is based primarily on hydrophobic differences in the membrane surfaces of the three preparations and is thus due to microsomal properties not previously utilized in their fractionation. The method permits additional subfractionations of microsomes.

Animals

Partition in two-polymer aqueous phases reflects differences between membrane surface properties of erythrocytes, ghosts and membrane vesicles.

Partition of cells (defined as the percentage of added cells in the top phase) in dextran-polyethylene glycol aqueous two-phase systems is an extremely sensitive indicator of cell surface properties. By appropriate choice of polymer concentrations and salt composition and concentration one can, to some extent, select the membrane properties (e.g. charge (Walter, H. (1975) in Methods in Cell Biology (Prescott, D.M., ed.), Vol. 9, pp. 25-50, Academic Press, New York), lipid composition (Walter, H, Krob, E.J. and Brooks, D.E. (1976) Biochemistry, 15, 2959-2964)) that determine the partition behavior of cells (or membranes). In the present experiments we have obtained by partition information on separability, alteration and ensuing heterogeneity during preparation of human erythrocytes, ghosts and rightside-out and inside-out vesicles from human erythrocyte membranes. A phase system in which partition is greatly (although not exclusively) dtermined by membrane charge was used. It was found that: 1. The partition coefficients (and hence the membrane surfaces) of ghosts and of rightside-out vesicles differ from those of the erythrocytes from which they are derived. 2. Rightside-out and inside-out vesicles have different countercurrent distribution patterns with the rightside-out vesicles having the higher partition coefficient (probably reflecting higher membrane charge of the latter). 3. Rightside-out vesicles are highly heterogeneous membrane populations as evidenced by broader than theoretical countercurrent distribution curve which, in some experiments, even split into two populations outright. This heterogeneity is particularly interesting since the enzyme markers used to establish vesicle sidedness (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed), Vol. 2, pp. 245-281, Plenum Press, New York) indicate "pure" rightside-out preparations. 4. Inside-out vesicles could, under no conditions tested, be rendered totally free of rightside-out vesicles according to the enzyme markers used to indicate vesicle sidedness (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed), Vol. 2, pp. 245-281, Plenum Press, New York). A discussion of factors involved in the partition of the vesicles and ghosts together with analogies to the partition behavior of stored or in vitro modified human red blood cells is presented.

Acetylcholinesterase