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.

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.

The "instantaneous" deformation of cartilage: effects of collagen fiber orientation and osmotic stress.

The present study was undertaken with two objectives in view. The first was to distinguish between the "instantaneous" deformation and creep of articular cartilage when subjected to a step loading in unconfined compression. This was done by observing changes in the specimen's diameter rather than its thickness. The second objective was to investigate experimentally the anisotropic behaviour of cartilage in a compressive loading mode, corresponding to the physiological situation. An apparatus was thus developed and constructed which enabled us to follow the "instantaneous" changes of the surface area of the sample as the latter was being loaded in unconfined compression. Specimens of human articular cartilage from normal femoral heads and condyles were tested. Full thickness specimens were tested with and without the underlying bone, as well as partial thickness specimens, characterizing the different zones of cartilage. Solutions of different ionic strength were used to vary the osmotic stress and specimens covering a considerable range of proteoglycan concentrations were selected. The effects of hydration and proteoglycan removal on the "instantaneous" deformation were also studied. The "instantaneous" deformation was found to be of a strongly anisotropic nature in all zones. The deformation was always smaller along the Indian-ink prick pattern than at 90 degrees to it, and this effect was most pronounced in the superficial zone of cartilage. The results reveal an analogy with the tensile properties of cartilage and indicate that the collagen network is mainly responsible for controlling the "instantaneous" deformation. The proteoglycans play an indirect role by modulating the stiffness of the collagen network through their osmotic pressure.

Contribution of NMDA and non-NMDA receptors to synaptic transmission from the brachium of the inferior colliculus to the medial subdivision of the medial geniculate nucleus in the rabbit.

Previous work from this laboratory has demonstrated that monosynaptic inputs from the brachium of the inferior colliculus (BIC) to the medial subdivision of the medial geniculate nucleus (mMG) strengthen as a result of associative conditioning with an acoustic conditioned stimulus (i.e., fear conditioning). One model that has been proposed to underlie certain types of neuronal plasticity involves the recruitment of N-methyl-D-aspartic acid (NMDA)-type glutamate receptors. The purpose of the present study was to examine the relative contributions of glutamatergic NMDA and non-NMDA receptors to synaptic transmission within this pathway. Individual contributions of the specific receptor types were assessed through the use of 2-amino-5-phosphonovaleric acid (AP5), a selective NMDA receptor antagonist, and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist. Bipolar stimulating electrodes were stereotaxically implanted in BIC and recording electrodes (attached to dual 32-gauge cannulae for delivery of drug) were positioned in mMG of New Zealand albino rabbits. Single pulses (150 micros, 100-350 microA) delivered to BIC resulted in short-latency (<4 ms) responses in mMG. BIC-evoked single-unit activity was recorded from mMG before, during, and at several intervals after injection of AP5, CNQX, and/or artificial cerebrospinal fluid (ACSF). Injection of either AP5 or CNQX, but not ACSF, significantly attenuated the short-latency BIC-evoked responses in the vast majority of cells tested. These findings suggest that the monosynaptic pathway from BIC to mMG is glutamatergic and that this pathway frequently employs NMDA-type receptors during electrically stimulated synaptic transmission. Due to the NMDA receptors' proposed role in plasticity (e.g., long-term potentiation), these results may have implications for understanding the mechanisms of synaptic plasticity observed at this synapse during associative learning.

Heterogeneity in the surface properties of B16 melanoma cells from sublines with differing metastatic potential detected via two-polymer aqueous-phase partition.

When mixed in aqueous solution at low concentrations, the neutral polymers dextran and poly(ethylene glycol) (PEG) rapidly form a two-phase system, consisting of a dextran-enriched lower phase and a PEG-enriched upper phase. Two B16 mouse melanoma cell lines, B16-F1 (low lung colonizing capability) and B16-F10 (high lung colonizing capability) were found to partition differentially into the upper phase in a variety of two-phase systems. Upper-phase partition depends primarily on either hydrophilic (i.e., surface charge density) or hydrophobic (i.e., affinity for the hydrocarbon chain of a PEG-fatty acid ester) cell surface properties, depending on the system used. In single-step partition studies, cells of the B16-F10 subline displayed a greater preference than B16-F1 cells for the upper phase in the hydrophilic system and less preference in systems sensitive to hydrophobic properties. Countercurrent distribution (CCD) experiments, performed with [125I]deoxyuridine DNA-labelled cells, were consistent with single-step partition results. These CCD results demonstrated that B16-F10 cells exhibited greater DNA synthesis than B16-F1 cells and that considerable heterogeneity, in both hydrophobic and hydrophilic surface properties, was present in subpopulations of cells of both sublines. The data also showed considerable enrichment of 125I-specific cell activity in certain sections of the distributions, indicating that differences in cellular DNA synthesis are reflected in the surface properties to which partition is sensitive.

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.