Harry Walter

Differential electrophoretic behavior in aqueous polymer solutions of red blood cells from Alzheimer patients and from normal individuals

The recently reported phenomenon that red blood cells (RBC) from Alzheimer disease (AD) patients and normal individuals, which have identical electrophoretic mobilities (EPM) in phosphate-buffered saline (PBS), have different EPM in appropriately selected polymer solutions, has been further explored. Of a number of in vitro treatments to which AD and normal RBC were subjected prior to EPM measurements in bottom phase (from a dextran-poly(ethylene glycol) (PEG) aqueous phase system) only trypsin eliminated the difference. Thus, the differential polymer interaction between AD and normal RBC, thought to be the basis for their dissimilar EPM, can be abolished by appropriate proteolytic modification of the cell surfaces and suggests protein as a source of difference. Because young and old RBC from normal individuals, which have the same EPM in PBS, have different EPM in certain polymer solutions, and the RBC from AD patients have been reported to age abnormally, we also compared the young and old RBC subpopulations from these two sources. By the criterion of cell electrophoresis in polymer solutions the differences between AD and normal RBC and between young and old RBC are distinct. The EPM of AD and normal RBC differ in bottom phase or PEG but not in dextran solution; while the EPM of young and old RBC differ predominantly in dextran. We speculate that since the observed difference in EPM of RBC from AD patients and normals depends on protein(s) yet is anticoagulant-related (being obtained only when blood is collected in citrate or oxalate) it might be the result of an interaction (Ca(2+)-mediated?) between the surfaces of these cells and protein component(s) of their respective, compositionally differing sera.

Immobilized metal ion affinity partitioning of erythrocytes from different species in dextranpoly(ethylene glycol) aqueous phase systems

Abstract Poly(ethylene glycol) (PEG)-bound ligands partition preferentially into the top, PEG-rich, phase of dextran (Dx)-PEG aqueous phase systems. The extraction of erythrocytes from beef, dog, horse, human, pig, rabbit, rat and sheep was examined in both non-charge-sensitive and charge-sensitive Dx-PEG phase systems containing PEG-iminodiacetate (IDA) which had been reacted with Cu(II) or Zn(II). PEG-IDA-Cu binds primarily to histidine (His) residues. Phase systems containing excess imidazole were used to obtain cell partition ratios not attributable to the metal chelate. In non-charge-sensitive phase systems having lower polymer concentrations a correlation has been reported between the partition, P , of erythrocytes and their membrane ratio of poly/monounsaturated fatty acids; while in charge-sensitive phases there is some correlation between the P values and the cells relative electrophoretic mobilities. At higher polymer concentrations red blood cells accumulate at the interface and do not partition. Under such conditions addition of the PEG-IDA-Cu (Zn is less effective) causes erythrocytes to partition into the PEG-rich phase in a non-charge-related or charge-associated sequence reminiscent of that found in the absence of chelate in non-charge-sensitive or charge-sensitive phase systems, respectively, at lower polymer concentrations. PEG-IDA-Cu may thus be useful in extending the partitioning range of Dx-PEG systems to cells having such low P values in non-charge-sensitive and/or charge-sensitive phase systems as to preclude their partitioning even when phase systems are optimized by manipulation of their components. From the cited experiments it would appear that either the His per unit surface area of erythrocytes from different species is about the same causing the non-charge-related or charge-associated surface properties (depending on the phase system used) to determine, to a large extent, the P even in the presence of chelate or that the non-charge-related or charge-associated surface properties outweigh the differences in His content and effect the observed correlations. In contrast to these apparently “non-specific” extractions effected by PEG-IDA-Cu, there are cases in which PEG-IDA-Cu acts as a sensitive probe for recognizing differences in cell surface properties not detected by other means.

Erythrocyte partitioning in dextran—poly(ethylene glycol) aqueous phase systems: Events in phase and cell separation

Early events in the partitioning process which involve characteristic kinetics of cell- and phase-specific interactions and phase separation have been described previously. This paper reports on red cell-phase droplet interactions pertaining at the time of usual phase sampling (i.e., the time at which a clear bulk interface is first apparent) and beyond in cell partitioning and countercurrent distribution experiments. In non-charge-sensitive phase systems close to the critical point, cells can be free or attached to phase droplets. Cells that are free are virtually completely in the top phase, whereas different cell populations that show essentially complete binding to droplets can nevertheless have different partition ratios and be separated, thus reflecting the effects of the difference in the cells avidity for the phase droplets during the early, elapsed events in partitioning. At higher polymer concentrations (i.e., higher interfacial tensions), the cell populations, completely bound to phase droplets, partition completely to the interface, and consequently cannot be separated. When such systems are made charge-sensitive by the generation of a Donnan potential between the phases or made into affinity systems by the incorporation of PEG ligands (e.g., PEG-palmitate), there is a decrease in the avidity of the cells for phase droplets. The resulting increase in the ratio of free to droplet-bound red cells in the top phase at the time of sampling correlates with an increase in the partition ratio, P, observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell partitioning in two-polymer aqueous phase systems and cell electrophoresis in aqueous polymer solutions red blood cells from different species

A correlation, with some exceptions, between the partitioning behavior of red blood cells (RBCs) from different species in charge-sensitive dextran-poly(ethylene glycol) (PEG) aqueous phase systems and their relative electrophoretic mobilities (EPMs) in phosphate-buffered saline (PBS) has previously been reported. This relationship has now been further probed by carrying out RBC electrophoresis in media (i.e., dextran-rich bottom or PEG-rich top phases) more closely approximating the environment in which RBC partitioning takes place to see whether a better correlation would ensue. The ratios of viscosity-corrected EPMs of different species RBCs in (diluted) dextran-rich or PEG-rich phases/EPMs of the respective species RBCs in PBS differ for a number of species, and from each other, reflecting thereby differences in kind (i.e., dextran or PEG) and nature of polymer interaction with these RBCs. There is a general tendency for EPMs in any of the tested media to correlate with both the cells relative partition ratios as well as with their relative EPMs in one of the other media. However, examination of the behavior of different species RBCs taken two species at a time indicates that their relative EPMs in any two suspending media or in one suspending medium and partitioning often differ. Thus, both the cell partition ratio and the cell EPMs obtained in polymer media must, at least in some cases, reflect surface properties other than or in addition to the charge reflected by EPM measurements in PBS or saline. Cell electrophoresis in polymer solutions thereby provides an additional parameter for discriminating between surface properties of certain closely related cell populations.

Conversion of serum proteins into tissue proteins.

Summary 1. Incorporation of S35 and C14 from injected doubly labeled rat serum albumin or globulin into the tissue proteins of rats was investigated. The ratio S35/C14 in tissue proteins varies very little during 9 days after injection of the doubly labeled serum proteins. 2. The ratio S35/C14 in tisues is approximately equal to (S35 P/C14 P) X (ST/SP) × Qc where the first term is the ratio of the 2 isotopes in the injected plasma protein, ST and SP the per cent of total sulfur in the analyzed tissue protein and the injected plasma protein, respectively, and Qc is approximately 1.5. 3. The increase in S35/C14 ratio (Qc > 1) indicates impoverishment of C14 relative to S35 and is attributed to utilization of C14-amino acids for other metabolic pathways. 4. In rats injected with serum albumin-S35, I131 the ratio S35/I131 increases during 9 days from 50 to 100 times. 5. Our results indicate that conversion of plasma proteins into tissue proteins involves breakdown to amino acids or very small peptide fragments. There is no indication for the transfer of large peptide fragments from plasma protein to tissue protein molecules.

Cell partitioning in two-polymer aqueous phase systems and cell electrophoresis in aqueous polymer solutions. Human and rat young and old red blood cells

It has recently been found that electrophoresis in solutions of appropriately selected polymers in phosphate-buffered saline (PBS) can differentiate between some closely related cell populations which have identical electrophoretic mobilities (EPM) in PBS (e.g., human young and old red blood cells (RBC); RBC from Alzheimer patients and normal individuals). The EPM differences detected in polymer solutions are most likely a consequence of cell- and polymer-specific interactions. Aspects of the relation between the electrophoresis in aqueous polymer solutions of native and in vitro treated young and old RBC (from human and rat) and their partitioning in a charge-sensitive dextran-poly(ethylene glycol) (PEG) aqueous phase system (i.e., a system with a Donnan potential between the phases, top phase positive) have been examined further and are discussed. Unlike the behavior of RBC from Alzheimer patients and normal individuals in which an EPM difference can be detected in PEG solutions but not in dextran, differences in EPM between human young and old RBC are detectable in solutions of either polymer. Selected enzyme treatments of human young and old RBC or their fixation with aldehyde eliminates the EPM differences in dextran; while neuraminidase treatment or formaldehyde fixation of rat young and old RBC retains EPM differences in dextran between these cells. In these latter cases partitioning differences are also in evidence and are in the same direction as the cells relative EPM (i.e., old RBC young RBC). Rat young and old RBC have different partitions (rat old RBC < young RBC) and different EPM (also rat old RBC < young RBC). Thus, while cell partitioning in a charge-sensitive dextran-PEG aqueous phase system and cell electrophoresis in polymer solution seem to reflect, at least with these cell subpopulations, qualitatively analogous differences in surface properties (in that increasing partitions and EPM are concomitant), there are instances in which either of these physical measurements discerns surface differences which escape detection by the other.

Effect of cell exposure to top or bottom phase prior to cell partitioning in dextran-poly(ethylene glycol) aqueous phase systems: erythrocytes as a model

Cells exposed to dextran (Dx)-rich bottom phase prior to cell partitioning in Dx-poly(ethylene glycol) (PEG) aqueous two-phase systems have lower partition ratios than cells exposed to PEG-rich top phase. Aspects of this previously observed phenomenon were explored. In the present work charge-sensitive phases made with Dx T500 and PEG 8000 were used exclusively. It was found that: (1) even on countercurrent distribution (CCD) red cells (RBC) loaded in bottom phase have a lower apparent partition ratio, G, than the same cells loaded in top phase; (2) when part of the same cell population is loaded into top phase and part into bottom phase of the same load cavities for CCD, with the cells loaded into top or bottom bearing an isotopic tracer (51Cr), the cells loaded into top phase have a higher G value than the cells loaded into bottom phase; (3) the shift in the CCD curves of human or of rat RBC between cells loaded in top or bottom phase using systems having the same polymer concentration (though different salt compositions) shows no striking difference and is, for the number of experiments run, not statistically significant; (4) when the quantity of cells loaded for CCD is reduced from 10(9) to 10(8), the G value of cells loaded in top phase is reduced slightly while that of cells loaded in bottom phase is diminished more appreciably; (5) increasing polymer concentrations yield larger differences in G values between (rat) RBC loaded in top or bottom phase; (6) when cells exposed to top or bottom phase, respectively, are centrifuged and suspended in bottom or top phase, respectively, their CCD patterns are qualitatively similar to cells exposed to these latter respective phases initially; (7) rat RBC populations containing 59Fe-labeled cells of different but distinct age are fractionated on CCD irrespective of whether loaded in top or bottom phase. An exception are populations containing very young mature labeled cells (e.g., 4-d old) which are resolved when loaded in top phase but not in bottom phase. Thus cell populations exist which can be resolved by CCD when loaded in one of the phases but not when loaded in the other. Glutaraldehyde-fixed rat RBC containing 4-d old labeled cells are fractionated by CCD irrespective of whether loaded in top or bottom phase.

Partitioning of cells in dextran-poly(ethylene glycol) aqueous phase systems : a study of settling time, vessel geometry and sedimentation effects on the efficiency of separation

The effect of prolonged settling times (up to 2 h), in high- and low-phase columns, on the cell partition ratios measured and on the separability of cell populations was examined. With closely related cell populations, modelled by rat erythrocytes in which subpopulations of red blood cells of distinct age were labeled isotopically, it was found that partitioning proceeds over the entire time period examined as evidenced by the continuous change in relative specific activity of cells in the top phase as the partition ratio falls. In control cell sedimentation experiments in top phase there was almost no change in the quantity of cells present when vertical settling (i.e., high-phase columns) was used and no separation of specific subpopulations was found. In the horizontal settling mode the initially higher cell partition ratio, as compared to vertical settling, decreased to a greater extent with longer time intervals; a given purity of cells only being obtained at a lower partition ratio than in the vertical settling mode. Cell sedimentation in top phase was appreciable with time in the horizontal settling mode but did not result in a separation of cell subpopulations. The effect of relative cell partition ratios and sizes in high- and low-phase columns on the efficiency of separation was examined by use of rat or sheep 51Cr-labeled red cells mixed with an excess of human unlabeled erythrocytes. Rat and sheep red cells are appreciably smaller than human erythrocytes. Rat red cells have higher, and sheep red cells lower partition ratios than human erythrocytes. With vertical settling, over a 2-h period, there is no appreciable contribution to the change in relative specific activities by cell sedimentation. However, the more rapid sedimentation of the larger human red cells has, with time, a measurable effect on the relative specific activities obtained during cell partitioning when run in the horizontal mode: enhancing the rat-human and diminishing the sheep-human cell separations. Partitioning cells in high-phase columns is of advantage with respect to increasing separation efficiency and virtually eliminating the influence of other physical parameters (e.g., cell size). Since the cell partitioning process continues for long periods of time, yielding ever-lower partition ratios with increasing proportions of cells with higher P values, a time may be selected which balances desired relative cell purity and yield.

Stability of an Azoprotein Hapten in the Organism.

Summary S35-sulfanilic acid was diazotized, coupled to beef serum γ-globulin and injected into rabbits. Four weeks after injection the animals were sacrificed. The liver protein, containing approximately 0.9% of the injected radioactivity, was hydrolyzed; cystine and methionine were isolated and found to contain only about 3% of the total radioactivity. Most of the activity was associated with a fraction which on chromatography behaved like an azo dye prepared from tyrosine and diazotized sulfanilic acid. It is concluded that azophenylsulfonate groups persist either unchanged or as slightly changed derivatives in the tissues over periods of more than one month.

Surface properties of lymphocyte subpopulations in autoimmune NZB/NZW F1 hybrid mice: Alterations correlated with the immunodeficiency of aging

Partitioning in a two-polymer aqueous phase system was used to probe the surface properties of lymphoid cell subpopulations in aged male NZB/NZW F1 hybrid (B/W) mice, an important model of autoimmunity, immunodeficiency, and lymphoid malignancy. Spleen cells were fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged dextran-polyethylene glycol system. CCD of spleen cells from young, clinically normal male B/W mice yielded several broad distribution patterns which frequently had two or more peaks. Analysis of differentiation antigens and functional properties of cells from different parts of the distribution revealed a subfractionation of the three major lymphocyte subpopulations. B lymphocytes had a low partition coefficient (K); T cells had an intermediate K and null cells had the highest K. To examine the partitioning behavior of T lymphocytes, spleen cells which were nonadherent to nylon wool columns were subjected to CCD. Nonadherent cells from young B/W mice consistently gave a single peak with high K. Aged mice (18 months) usually had nonadherent cells with a predominantly low K. In some experiments a systematic increase in the number of these cells could be demonstrated with increasing mouse age. An analysis of the adherence and partitioning behavior of lymphocyte subpopulations revealed no change in the adherence properties or proportions of B lymphocytes in aged mice. The large proportion of cells having a low partition coefficient in the nonadherent spleen cell population of old mice appears to be due to an increase in the number of null cells and in a decrease in the K of some T lymphocytes.