Lysis

About: Lysis is a research topic. Over the lifetime, 6072 publications have been published within this topic receiving 216978 citations.

Cytotoxic activity of human lymphocytes: quanitative analysis of T cell and K cell cytotoxicity, revealing enzyme-like kinetics

Abstract: The mechanism of action of human cytotoxic lymphocytes was studied in two systems: T cell cytotoxicity in cell-mediated lympholysis (CML) and K cell cytotoxicity in antibody-dependent lymphocytotoxicity (ADL). The mechanisms resemble that of an enzyme, if the effector cells are regarded as enzyme and the target cells as substrate. On the basis of a kinetic analysis it was shown that the experimental data are compatible with a simple rate equation, which allows the definition of three constants: p , representing the maximal killing rate of the effector cell suspension (at target →∞); q , representing the maximal lysis of the target cells (at effector →∞); K which represents the Michaelis constant (i.e, the target cell number giving ½ V max ), when extrapolated to effector = 0. The constant K may be taken to reflect the affinity between effector and target cells. Furthermore, for the ADL system the influence of the effector cell concentration on Km was due not to the addition of inert cells but rather to the presence of cells that can bind but not kill. Finally, it was shown that in the ADL a K cell can recycle, i.e., kill more than one target cell. On the basis of these results, experimental conditions are proposed for optimal measurement of cellular cytotoxicity.

Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes.

Abstract: While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.

A simple, efficient method for the separate isolation of RNA and DNA from the same cells.

Abstract: A method for the isolation of total cytoplasmic RNA and high molecular weight DNA from the same cells is described. Cells are gently lysed with NP40 in the presence of vanadyl ribonucleoside complex and the nuclei pelleted by centrifugation. RNA is purified by phenol/CHCl3 extraction of the lysate supernatant followed by ethanol precipitation. Protein is removed from high molecular weight DNA by salt-precipitation after nuclei are digested with proteinase K in the presence of sodium dodecyl sulfate. High yields of clean, intact RNA and DNA are obtained. A major advantage of the method is that it can be scaled down to quantitatively extract RNA and DNA from as little as 1000 cells.