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E J Krob

Publications and source records attributed to E J Krob.

At least 37 records · Page 2Linked to original sources
PubMed1982

Subfractionation of cell populations by partitioning in dextran-poly (ethylene glycol) aqueous phases. "Discriminating" and "nondiscriminating" systems.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous two-phase systems depends on the interaction between the surface properties of the cells and the physical properties of the phases. The latter can be manipulated to a considerable extent by selection of polymer concentrations and ionic composition and concentration. If salts (e.g., phosphate) are used that have an unequal affinity for the two phases, an electrostatic potential difference between the phases results and, at appropriately high polymer concentrations, the partition coefficient of cells is determined predominantly by membrane charge-associated properties. By "balancing" the magnitude of the electrostatic potential difference against that of the interfacial tension (primarily a function of polymer, but also phosphate, concentrations) one can obtain phase systems that give usable partition coefficients for most cell populations (1). In work under way in our laboratory on the effects of different chemical and enzymatic modifications on the relative surface properties of rat red blood cells of different ages, we have now found that certain phase compositions did not resolve such treated cell subpopulations while other phase compositions did. Thus not all charged phase systems in which cell populations as a whole have usable partition coefficients are equally capable of detecting or subfractionating cell subpopulations. It is therefore essential, before drawing conclusions on the nonseparability of cell subpopulations, to test cell separability in charged phase systems of different compositions if the system initially chosen does not afford a subfractionation.

Age Factors↗
PubMed1981

Aging of erythrocytes results in altered red cell surface properties in the rat, but not in the human. Studies by partitioning in two-polymer aqueous phase systems.

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.

Animals↗
PubMed1980

Correlation between phagocytic and membrane surface properties reflected by partitioning of human peripheral blood monocytes in two-polymer aqueous phases.

Human peripheral blood monocyte-enriched fractions (identified by staining for peroxidase and by sizing) were obtained by velocity sedimentation at unit gravity of peripheral blood mononuclear cells. They were then fractionated by countercurrent distribution (a multiple-extraction procedure) in a charged Dextran/poly(ethylene glycol) aqueous phase system. The monocytes remained viable after the separation (order of 90%). Cells obtained from different cavities along the extraction train were tested for their ability to phagocytize latex particles. With increasing partition coefficient (presumably higher charge-associated membrane properties) the ratio of monocytes that phagocytized to monocytes that did not phagocytize increased appreciably. When, however, monocytes were permitted to phagocytize particles prior to countercurrent distribution, an increase in partition coefficient was associated with an appreciable decrease in the above-specified ratio. Control experiments indicate that the observed change in partitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitiartitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitioning) and their ability to ingest particles. Furthermore, an alteration in the surface charge-associated properties of monocytes as a consequence of phagocytosis is indicated by the cells' reduced partition coefficient.

Cell Membrane↗
PubMed1979

Effect of membrane cholesterol enrichment or depletion on the partition behavior of human erythrocytes in dextran-poly(ethylene glycol) aqueous phases.

It has previously been shown that by appropriate manipulation of polymer concentrations and ionic composition and concentration one can select whether charge-associated or lipid-related membrane surface properties are reflected by cell partition in dextran-poly(ethylene glycol) aqueous two-phase systems (Walter, H. (1977) in Methods of Cell Separation ((Catsimpoolas, N., ed.), Vol. 1, pp. 307-354, Plenum Press, New York). In the current experiments we have studied that partition behavior of human erythrocytes and found that not only lipid-related but also charge-associated membrane properties are altered as a consequence of cholesterol-enrichment or -depletion. Results further indicate that, just as cell partition in charged phase systems reflects membrane charge-associated properties not readily measured by means other than partition (Brooks, D.E., Seaman, G.V.F. and Walter, H. (1971) Nat. New Biol. 234, 61--62; Walter, H., Tung, R., Jackson, L.J. and Seaman, G.V.F. (1972) Biochem. Biophys. Res. Commun. 48, 565--571), cell partition in uncharged phases reflects membrane lipid-related properties also not readily measured by other means.

Cell Separation↗
PubMed1979

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↗
PubMed1979

Subfractionation of human peripheral blood lymphocytes on the basis of their surface properties by partitioning in two-polymer aqueous phase systems.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous-aqueous two-phase systems is a sensitive method for separating cells and for obtaining information on their surface properties. Highly purified lymphocytes were obtained by velocity sedimentation of human peripheral blood mononuclear cells and fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged two-polymer aqueous phase system. The lymphocytes remained viable after separation (order of 90%) and the E-rosetting cells responded (after adding back monocytes) to mitogens (PHA, Con A, PWM). Not only was the total lymphocyte population found to be highly heterogeneous (as evidenced by a broad and skewed distribution curve), but we were able to show that cells that rosetted with E, or had complement or Fc receptors were composed of additional subpopulations as well. The bulk of complement-receptor-bearing cells had the lowest partition coefficient (K), E-rosetting cells an intermediate K, and Fc-receptor-containing cells the highest K. The largest lymphocytes were among the subpopulation having the highest K and neither responded to T cell mitogens nor rosetted with E. Our results thus demonstrate that human peripheral blood lymphocytes can be subfractionated by CCD. The fractions are differentially enriched with lymphocyte subpopulations having characteristic surface markers and functional abilities.

Animals↗
PubMed1976

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↗
PubMed1976

Membrane surface properties other than charge involved in cell separation by partition in polymer, aqueous two-phase systems.

When aqueous solutions of dextran and of poly-(ethylene glycol) are mixed above certain concentrations, immiscible, liquid two-phase systems are obtained which are useful for separating cells by partition. Some salts partition unequally between the phases, giving rise to an electrostatic potential difference between them. Partition of cells has therefore been thought to depend predominantly on membrane charge. We now report two instances in which membrane charge either does not determine or is not the main determinant of cell partition. (A) Cell partition coefficients in phase systems approaching the critical point (the component concentrations below which a homogeneous solution occurs) increase, even in phase systems in which the phase potential difference is practically zero. Furthermore, in such systems, the partition coefficient of (human) erythrocytes is not reduced by complete removal of sialic acid. (B) Rat and mouse erythrocytes have sizable partition coefficients in a phase system away from the critical point with no potential difference between the phases. Cell surface interaction with the polymers is probably responsible for cell partition in these cases. Partition studies on erythrocytes from nine mammalian species in phases near the critical point with and without electrostatic potential differences reveal major species-specific differences in the membrane charge/noncharge components. A correlation has been found, in phases near the critical point that have essentially no electrostatic potential difference, between partition coefficient and the ratio of poly/monounsaturated fatty acids in the membranes of red cells from different species. Our present results thus provide parameters for the separation of cells by partition in addition to or instead of membrane charge depending on the polymer and salt composition and concentration selected.

Animals↗
PubMed1975

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↗