Electrical properties of cell pellets and cell electrofusion in a centrifuge.

A new approach is proposed for studying cell deformability by centrifugal force, electrical properties of cell membranes in a high electric field, and for performing efficient cell electrofusion. Suspensions of cells (L929 and four other cell types examined) are centrifuged in special chambers, thus forming compact cell pellets in the gap between the electrodes. The setup allows measurement of the pellet resistance and also the high-voltage pulse application during centrifugation. The pellet resistance increases sharply with the centripetal acceleration, which correlates with reduction of the cell pellet porosity due to cell compression and deformation. Experiments with cells pretreated with cytochalasin B or colcemid showed that cell deformability depends significantly on the state of cytoskeleton. When the voltage applied to the cell pellet exceeds a 'critical' value, electrical breakdown (poration) of cell membranes occurs. This is seen as a deflection in the I(V) curve for the cell pellet. The electropores formed during the breakdown reseal in several stages: the fastest takes 0.5-1 ms while the whole process completes in minutes. A novel effect of colloid-osmotic compression of cell pellets after electric cell permeabilization is described. Supercritical pulse application to the cell pellet during intensive centrifugation leads to massive cell fusion. The fusion index grows with the increase of centripetal acceleration, and drops drastically when the pulse is applied after the centrifuge is stopped. The colloid-osmotic pellet compression enhances the fusion efficiency. No fusion occurs when cells are brought in contact after the pulse treatment. The data suggest that tight intermembrane contact formed prior to pulse application is a prerequisite condition for efficient cell electrofusion. The capacities of the technique proposed and the mechanism of membrane electrofusion are discussed.

Electrofusion of fibroblasts on the porous membrane.

Electric fusion of cells is usually performed in two steps: the first is the creation of tight intercellular contact, the second is an application of electric pulses which induce membrane fusion proper. In the present work a new technique of cell electrofusion on the porous film is described. It consists of preliminary cultivation of cell monolayer on the porous film (protein-coated cellophane). Then cells of the same or any other type are added from above to form a second cell layer upon the first one. The pulses of the electric field are applied normally to the plane of the double cell layer to induce cell fusion. After pulse application a picture of mass polynucleation was observed. At the same time we did not obtain fusion of L cells by means of dielectrophoretic electrofusion technique. This difference in efficiency could be explained by the formation of broad zones of membrane contact between the cells adherent to the film, while during intensive dielectrophoresis only the point contacts were revealed. The high-conducting medium for electric treatment providing an efficient fusion on the film and high cell viability was composed. Neither cytochalasin B nor colcemid affected cell fusion noticeably; however the sodium azide (added with 2-deoxyglucose) inhibited fusion completely. The short hypotonic shock after electric treatment enhanced the rate of polycaryon formation.

[Electron microscope study of chromatin in hepatocyte nuclei during the first hours after partial hepatectomy. V. Changes in the relative area of condensed chromatin and the density of chromatin fibril packing in the ultrathin sections]. / Elektronno-mikroskopicheskoe izuchenie khromatina v iadrakh gepatotsitov v pervye chasy posle chastichnoi gepatéktomii. V. Izmenenie otnositel'noi ploshchadi kondensirovannogo khromatina i plotnosti upakovki fibrill na ul'tratonkikh srezakh.

The degree of chromatin condensation was studied on ultrathin cell sections of guinea pig hepatocytes during the prereplicative period after partial hepatectomy. Three time points were chosen for analysis namely 2,5, 5 and 9 hrs after operation since they show marked increasing (2.5 hrs), decreasing (5 hrs) and repeated increasing (9 hrs) of the amount of ethidium bromide binding to chromatin. The degree of chromatin condensation was determined by measuring the area occupied by condensed chromatin and also by measuring the number of chromatin fibrils per a certain length. The condensed chromatin with varying localization in the nucleus were studied separately. The changes of nucleoplasmic chromatin were most pronounced: at 2.5 and 9 hrs after operation the decrease of the relative area and of the density of chromatin fibrils package was observed; these parameters were near to control at 5 hrs after operation. In general the changes in nucleoplasmic chromatin were correlated with the changes of the activity of the chromatin in the whole nucleus. The decondensation of the perimembranous chromatin was manifested in the decrease of its area and was expressed only at 9 hrs after operation. The perinucleolar chromatin was found to show the gradual decondensation which was manifested mainly by the decrease of its relative area. Thus the condensed chromatin seems to be a labile structure which undergoes essential changes in the process of the exit of the hepatocytes from G0-stage of the cell cycle, during the prereplicative period.

[Electron microscopic examination of hepatocyte nuclei within first hours after partial hepatectomy. III. Changes of DNP fiber diameters of condensed chromatin]. / Elektronno-mikroskopicheskoe izuchenie khromatina v iadrakh gepatotsitov v perbye chasy posle chastichnoi gepatektomii. III. Izmenenie tolshchiny DNP-fibrill v sostave kondensirovannogo khromatina.

The DNP fibers have been measured on electron microscope photographs of sections of guinea-pig hepatocytes. The condensed chromatin was found to have fibers from 4 to 27 nm in diameter. Within the first hours after partial hepatectomy the distribution of fibers by their diameters is changed. This is correlated with the ethidium bromide binding to chromatin DNP, which reflects the level of the chromatin template activity. Maximal binding to ethidium bromide was observed at 2.5 hours after the operation and the distribution of fiber diameters was shifted towards a smaller diameter, simultaneously, the amount of thinner fibers from 4 to 14 nm in diameter is larger. Five hours after the operation the content of thicker fibers from 14 to 24 nm is reduced, and the intensity of the ethidium bromide binding is decreased. Nine hours later, the ethidium bromide binding was intensified, and the distribution of fiber diameters again shifted to a smaller width. Our data prove that the thickness of DNP fibers of condensed chromatin, which has been thought inactive, depends on the intensity of the template synthesis.

Electron microscopic studies of guinea pig hepatocyte chromatin.

Chromatin from guinea pig hepatocytes has been studied by electron microscopy using the spreading technique. Among DNP fibers with nucleosomal organization numerous structures with different organization have been observed. The structures consist of the main axial fiber from which the lateral fibrils branch off normal to the axis. The distances between the bases of lateral fibrils vary to a considerable extent. The lateral fibrils are composed of regular granules with an average diameter of 257 A, and the axial fiber of irregularly and rarely arranged granules. The structures described are thought to be nonribosomal transcription complexes, and the granules of lateral fibrils--nuclear informosomes. The longest lateral fibril may have up to 80 granules (informosomes), which corresponds to a pre-mRNA chain of about 15-17 x 10(6) daltons.