Colloidal Silica Density Gradient Research Articles

De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus alpha-toxin. Inhibition by nonhydrolyzable GTP analogs.

The role of GTP-binding proteins in autophagic vacuole formation was investigated in isolated rat hepatocytes permeabilized by alpha-toxin from Staphylococcus aureus, an agent which creates stable plasma membrane channels allowing exchange of small (< or = 1000 Da) molecules. Vacuole formation was monitored from the uptake of 125I-tyramine-cellobiitol (125ITC) into osmotically sensitive vacuoles isolated on colloidal silica density gradients. Separation was based on an established observation that autophagic vacuoles are retained in a heavy midgradient band when samples are layered, but are selectively shifted to dense fractions when they are previously dispersed in the gradient material. The vacuolar uptake of 125ITC was concentration-dependent and required exogenous ATP: 94% was directly mediated by sequestration; 6% was acquired by fluid-phase endocytosis as monitored by [carboxyl-14C]dextran-carboxyl. Although the amino acid control of proteolysis was lost, addition of the nonhydrolyzable GTP analog GTP gamma S (as well as GMP-PNP) decreased fractional rates of direct vacuolar 125ITC uptake and long-lived proteolysis by similar amounts (1.02-1.03% h-1), substantiating the notion that the effects were the direct result of autophagic inhibition. These and associated findings, supported by quantitative electron microscopy, indicate the presence of ongoing macro- and microautophagy in alpha-toxin-permeabilized cells and suggest that one or more GTP-binding proteins is required in macroautophagic vacuole formation.

Purification of thyroid lysosomes by colloidal silica density gradient centrifugation.

A procedure was devised for fractionating crude thyroid lysosomal particles (P750-15,000) by self-forming density gradient centrifugation with colloidal silica. Two discrete particle-containing peaks were observed, based on 131I-labeling and acid phosphatase activity: a heavy peak (density, 1.11-1.12) and a light peak (density, 1.05). Ultrastructural analysis revealed that the heavy peak consisted almost entirely of lysosomes, whereas the light peak represented a heterogeneous mixture of small vesicles and fragments of other intracellular organelles. In thyroids removed from rats 30 min after 131I injection, almost all of the 131I was present in the low density peak. This 131I appeared on sucrose density gradient centrifugation as a 19S peak, and it was almost completely insoluble in trichloroacetic acid. This was interpreted as indicating that the low density peak contained pinocytotic vesicles. In thyroids removed 4 days after 131I injection, the radioactivity appeared largely in the high density peak. Both the trichloroacetic acid solubility and the pattern on sucrose density gradient centrifugation indicated that the [131I] thyroglobulin had undergone extensive proteolysis. Thyroglobulin proteolytic activity was found primarily in the high density particles and to only a small extent in the low density particles. Studies performed at intervals after 131I injection combined with double labeling (131I and 125I) experiments provided evidence that radioactivity was transferred from the low density to the high density particles. Heterogeneity existed within the dense peak, related to the degree of thyroglobulin degradation, as it was observed that thyroid lysosomes become denser with increasing proteolysis of thyroglobulin. The acid phosphatase in the low density particles could be distinguished from that in the high density (lysosomal) particles by its elution pattern on Sephadex G-200 column chromatography, its response to freezing and thawing, and its reactivity with p-nitrophenylphosphate. It was concluded, therefore, that the acid phosphatase in the low density fraction was derived from prolysosomal structures such as vesiculated Golgi-endoplasmic reticulum-lysosomes. The prolysosomal acid phosphatase associated with the low density fraction appeared to be a large membrane-bound molecule which could be transformed into lysosomal acid phosphatase by incubation at pH 5.0.

Reduction of Ca 2+ uptake induced by ionophore A23187 of red cells from malaria ( Plasmodium berghei)-infected mice

The Ca 2+ ionophore A23187 has much less capacity to induce Ca 2+ uptake of red cells from P. berghei-infected mice than of cells from normal mice. The reduction in Ca 2+ uptake occurs in both uninfacted and infected cells at all stages in the infected blood, as shown from experiments with cells separated on colloidal silica density gradient. Measurement of the ionophore concentration in the medium reveals that the ionophore is partitioned into red cells from infected blood to a greater extent than cells from normal blood. The reduction in A23187-induced Ca 2+ uptake may be due to difference in the interaction of red cell membrane with the ionophore, and its already high Ca 2+ permeability.

Separation of functional subpopulations of murine and human lymphoid cells on colloidal silica density gradients. I. Preparation of the Ludox AM ® gradient material and characterization of separation capacities

This report describes a unique modification of an isopycnic density gradient system utilizing as a separating menstrum colloidal silica (Ludox AM ®). The primary advantages of this preparation are: (1) It is chemically defined, allowing extremely reproducible cell separation employing different lots of material; (2) the physical parameters (pH, density, salt concentration) of the final gradient suspension can be manipulated over a wide range of values, allowing for the separation of many different biological materials; (3) it allows separation of very large numbers of lymphoid cells with greater than 95% recovery of applied cells; (4) separated cellular subpopulations can be easily washed free of silica and cellular function is retained. This paper is a report of the preparation and functional characteristics of the gradient material as it relates to the separation of very large numbers of lymphoid cell subpopulations in both mouse and man. Subpopulations of murine and human lymphocytes separated by this gradient material were assayed for IgM synthesis, T-cell mediated cytotoxicity, and lymphokine production.

Rapid separation of living cells by colloidal silica density gradient centrifugation

Rapid separation of living cells by colloidal silica density gradient centrifugation

Separation of HeLa cells by colloidal silica density gradient centrifugation. I. Separation and partial synchrony of mitotic cells.

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040-1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.

The purification of lysosomes from HeLa cells by centrifugation in colloidal silica density gradients

The development and use of colloidal silica density gradient media is described, in which the osmolarity, density and pH are easily adjusted and maintained. Gradients formed in zonal, angle-head and swinging-bucket rotors were used to obtain lysosome preparations free of mitochondria as observed enzymatically and in the electron microscope.