Adaptive properties of the cultured cells from a pig embryo kidney exposed to hypotonic media

A high tolerance of the cultured SPEV-cells was found under vital examination of their behaviour in hypotonic conditions. The dilution of cultured medium 1:1 hardly affects the cell morphology. With a 1:3 dilution, some minute alterations in the cells were noticed associated with the cell shape and the structure of the nucleus and of the cytoplasm, which later gradually disappear. A marked tendency of morphological alterations in the cells increases with increasing the medium dilution (1:7 and 1:15), however no cell destruction was observed. The complete lysis of the cells occurs only in the medium diluted by 32 times (1:31).     

Effect of cytochalasin D on DNA synthesis in cultured cells

The effect of cytochalasin D (CD), an agent specifically destroying actin cytoskeleton, on DNA replication of cultured mouse embryonic fibroblasts (MEF) and BALB/3T3 strain cells was studied. Incubation of normal cells with CD resulted in progressive inhibition of DNA synthesis: in the first 16-20 h the percentage of cells pulse-labelled with 3H-thymidine was similar to that in control cultures, on day 8 the percentage of labelled cells was 7-8 times lower than in the control. The transfer of cells into fresh medium upon 8-day incubation in the presence of CD resulted in the recovery of DNA synthesis. Similar curves of DNA synthesis inhibition in the presence of CD and of DNA synthesis recovery in fresh medium were observed both in mononuclear and binuclear cells. Thus, CD-induced reorganization of actin cytoskeleton can have an abrupt but reversible disturbing effect on normal cell cycle.     

Chloride transport in human fibroblasts is activated by hypotonic shock

Incubation of human skin fibroblasts in hypotonic media induced the activation of 36Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of 36Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also 36Cl- influx was enhanced by hypotonic medium.     

The isolation of protoplasts from the placental cells ofSolanum nigrum L.

Summary Living protoplasts were isolated from the interplacental regions ofSolanum nigrum berries by the removal of the walls from cells in tissue slices treated for 1–2 hours with 12% pectinase in 0.33 M to 0.38 M sucrose solution. Protoplasts thus isolated, then washed and transferred to microculture chambers for observation, invariably tended to be spherical. Comparative measurements of cell and protoplast volumes revealed that 10% of the isolated structures were subunits of protoplasts. From diameter changes in protoplasts studied in a hypotonic (0.20 M) sucrose solution, the maximum expansion of the plasma membrane was determined. Slightly hypertonic solutions (0.33 M to 0.38 M sucrose) promote stability of isolated protoplasts for several days. The importance to stability of osmotic concentration and ion balance in the medium is here established. Probably of equal importance is the optimal combination of several common constituents of culture media. Further studies on some aspects of specific medium requirements are in progress.This work was supported by a special grant from the Office of Advanced Studies and Research, University of South Carolina.     

Formation of inside-out vesicles of Bacillus licheniformis. Dependence on buffer composition and lysis procedure.

1. The extent to which the cytoplasmic membrane of the Gram-positive bacterium Bacillus licheniformis formed inside-out vesicles was studied with the freeze-fracture technique. The membrane orientation appeared to be dependent on the buffer compositon as well as on the lysis procedure used. 2. By manipulating these conditions, membrane preparations were obtained with the percentage of inside-out vesicles varying from 15 to 80%. 3. More vesicles had the opposite orientation when the cells were lysed in potassium phosphate buffer than when they were lysed in sodium phosphate buffer. Tris-HCl buffer favoured the formation of inside-out vesicles more than phosphate buffer. 4. Lysis of protoplasts in hypotonic buffers resulted in more inside-out vesicles than did direct lysis of cells in hypotonic media. 5. In an attempt to explain the observed differences, experiments were performed in which the morphology of thin-sectioned lysing cells in sodium phosphate buffer was compared with that in potassium phosphate buffer. The results from these experiments indicate that the formation of inside-out vesicles is brought about by an effect on the membrane itself rather than on the cell wall, on the cell wall membrane association, or on the cytoplasm.     

鸡胚与胎鼠的成纤维细胞条件培养液促进成肌细胞增殖和融合的比较

用培养过鸡胚(来亨鸡)或胎鼠(ICR小鼠)肌组织的成纤维细胞的条件培养液,定量地研究它们对胎鼠或鸡胚的成肌细胞的增殖和融合的影响。所得结果如下:(1) 胎鼠的成纤维细胞条件培养液促进胎鼠或鸡胚成肌细胞增殖,分别为对照组的2.65倍,(P<0.001)或2.35倍,(P<0.01);(2) 鸡胚的成纤维细胞条件培养液促进鸡胚或胎鼠的成肌细胞增殖,分别为对照组的2.66倍,(P<0.01)或2.17倍,(P<0.01);(3) 胎鼠的成纤维细胞条件培养液增加胎鼠或鸡胚的成肌细胞的融合率,分别为对照组的1.9倍或2.6倍;鸡胚的成纤维细胞条件培养液只增加鸡胚成肌细胞的融合率,为对照组的2.1倍,但对胎鼠成肌细胞的融合无明显的影响。 实验结果提示:成纤维细胞条件培养液促进成肌细胞的增殖,两种动物间无明显的差异,但在融合上却有一定的种属特异性。     

Functional evaluation of cultured cells under hypotonic conditions by intravital staining with neutral red

A high stability of pig embryo kidney-cells to hypotony was shown with neutral red vital staining. In the medium, diluted 1 : 1, the cells kept their ability to make granules during 6 days. In the medium diluted by water 1 : 3, both granular and diffuse staining of cells was shown within 6-12 hours. Later (18-48 hours) the cell ability to make granules was kept, but the neutral red was deposed as large vacuoles. In the normal medium the cell ability to make granules was kept too. In the medium, diluted by water 1 : 7, the cells lost their ability to make granules. The ability was recovered when cells were returned to the normal medium. Thus, after 6-18 hours of incubation in such a hypotonic medium, the cells produced small granules of neutral red, and 24-48 hours later they made large granules.     

Actin distribution in somatic embryos and embryogenic protoplasts of white spruce (Picea glauca)

Summary Examination of unfixed immature somatic embryos of white spruce (Picea glauca) with fluorescent rhodamine-labeled phalloidin revealed an extensive network of fine actin microfilaments (MFs) in the embryonal region which were not detected in specimens fixed with formaldehyde. Transition cells linking the embryonal region and suspensor cells contained fine MFs as well as bundles of MFs. The large, highly vacuolated suspensor cells were characterized by actin MF cables only. Treatment of embryos with cytochalasin B (CB) removed the fine MFs from the embryonal region and transition cells, but many MF cables in suspensor cells were resistant. Full recovery from CB treatment was observed in most somatic embryos. Embryogenic protoplasts capable of regenerating to somatic embryos in culture were released from only the embryonal region of somatic embryos. Both uninucleate and multinucleate embryogenic protoplasts retained the extensive network of fine actin MFs. In contrast, protoplasts derived from vacuolated suspensor cells and vacuolated free-floating cells contained thick MF bundles and were not embryogenic. Distinct MF cages enclosed nuclei in multinucleate protoplasts and may be responsible for preventing nuclear fusion. Microspectrophotometric analyses showed that the DNA contents of embryonal cells in the embryo and embryogenic protoplasts were similar and characteristic of rapidly dividing cell populations. However, transition and suspensor cells which released nonembryogenic protoplasts appeared to be arrested in G₁, and suspensor cells showed signs of DNA degradation.     

The role of the cytoskeletal elements during the return to the cell center of their nuclei displaced by centrifugation]

Living cells of the monolayer cultures of embryonal pig kidney epithelium (PKE-cells) and of embryonal bovine tracheal cells (FBT-cells) were ultracentrifuged at 20,000g. The centrifugal force was directed parallel to the surface of the culture slides. Just after centrifugation, the cellular nuclei were displaced to the centrifugal parts of cells. Centrifuged slides with cells were returned to the normal culture conditions, and 22 h later the nuclei were seen to restore their central position in the cells. The motion of the nuclei to the cell center was rather chaotic both in direction and speed. The speed of this motion never exceeded several microns per hour. After nocodasole treatment (0.1-10 mkg/ml) of the cells or in a hypotonic medium, the distance of nuclear dislocation during centrifugation was longer, and the nuclei returned to the cell centers faster than in the control ones. After cytochalasin B treatment (2 mkg/ml), the nuclei moved to the cell centers somewhat more slowly than they did in the control cells. Thus, the establishment of the central position of nuclei in the cells takes place in the absence of microtubules or intermediate filaments. Probably, the central position of nuclei depends mainly on the action part of the cytoskeleton.     

Disparate aggrecan gene expression in chondrocytes subjected to hypotonic and hypertonic loading in 2D and 3D culture

The effects of hypotonic (180 mOsm) and hypertonic (580 mOsm) medium loading on chondrocyte aggrecan gene expression in 2D monolayer and 3D hydrogel culture (agarose or alginate) were studied. Aggrecan promoter activity was monitored using a luciferase reporter gene assay and transient transfection. Osmotic loading was observed to differentially affect promoter activity, with hypotonic loading generally producing at least a 40% elevation in promoter activity, except for the case of alginate where a 50% suppression was observed. Hypertonic loading produced at least a 35% decrease in activity for all cultures. Similar osmolality-induced changes to aggrecan mRNA levels were observed in monolayer cells using qPCR. Deletion of exon 1 blocked the sensitivity of monolayer cells to hypertonic but not hypotonic medium changes. Confocal microscopy measurements suggested that the degree of hypotonic swelling in cells encapsulated in 3D matrix was restricted compared to monolayer cells whereas the degree of hypertonic shrinking was similar under both culture conditions.     

The actin cytoskeleton is involved in signalling protoplast embryogenesis induced by agarose embedding

The organization of actin microfilaments was studied by immunofluorescence in protoplasts isolated from sunflower hypocotyls and cultured in an agarose matrix. Removal of the cell wall completely disrupted the actin cytoskeleton, which became progressively reorganized into cortical microfilament arrays and actin cables during protoplast culture. Treatment of protoplasts with arginine-glycine-aspartic acid (Arg-Gly-Asp) motif-containing peptides, to inhibit putative cell contacts with the agarose matrix, strongly affected this repair process: microfilament elongation and cable formation were inhibited and the connectivity between the cortical network and the perinuclear basket was lost. Furthermore, embryoid formation induced by agarose embedding was reduced. Similar effects were observed with a short treatment with latrunculin B, known to disrupt actin microfilaments. These results indicate that the actin network is involved in the signalling process that leads to polarity acquisition and embryoid determination in agarose-embedded protoplasts.     

Optimum conditions for the oxidation of palmityl-carnitine by the mitochondria of rat brown adipose tissue.

The optimum conditions for maximum oxidation of palmityl-carnitine by mitochondria isolated from the brown adipose tissue of 10-day-old rats was studied. The findings were as follows: 1. Reduction of the sucrose osmolar concentration to below 100 mM activates the rate of palmityl-carnitine oxidation, the maximum effect being achieved with 25 mM sucrose. These hypotonic conditions lead to enlargement of the matrix compartment, but not to swelling of the whole mitochondria. The maximum respiration rate in 25 mM sucrose can be measured only with fresh mitochondria isolated less than 1 hour previously, which must be preincubated 5 minutes in hypotonic sucrose before adding palmityl-carnitine. 2. When inducing the maximum palmityl-carnitine oxidation rate in 100 mM KC1 medium the preincubation time must be prolonged to at least 8 minutes. The length of time for which the mitochondria are stored in isotonic sucrose at 0 degrees C does not affect the respiration level in the presence of K+ ions. 3. The optimum palmityl-carnitine concentration is the same for oxidation measured in hypotonic sucrose and in KC1 medium and ranges from 15 to 50 muM. 4. If the above conditions are observed, the maximum palmityl-carnitine respiration values in hypotonic sucrose and medium with K+ ions are the same, whereas in isotonic sucrose respiration is inhibited. The same applies to the oxidation of endogenous fatty acids by the carnitine route and to alpha-ketoglutarate respiration, while the oxidation of alpha-glycerolphosphate is not affected by the osmotic conditions and its respiration is the same in both hypotonic and isotonic sucrose media.     

Medium conditioned by feeder cells inhibits the differentiation of embryonal carcinoma cultures

Non-dividing STO mouse fibroblasts have been used for some time as feeder cells for maintaining certain embryonal carcinoma cells in an undifferentiated state. We report here that medium conditioned by these feeders can inhibit embryonal carcinoma (ec) cell differentiation induced either by removal from feeders, or, in the case of cells not normally requiring a feeder layer, by retinoic acid treatment.     

Osmotic Stress Alters the Intracellular Distribution of Non-erythroidal Spectrin (Fodrin) in Bovine Aortic Endothelial Cells

Cell swelling is known to result in unfolding of membrane invaginations and restructuring of F-actin. The effect of cell swelling on the intracellular distributions of other cytoskeletal proteins that constitute the submembrane cortical cytoskeleton is virtually unknown. This study focuses on the effects of cell swelling on non-erythroidal spectrin (fodrin, also known as spectrin II), a predominant component of the membrane cytoskeleton. The intracellular distribution of spectrin in vascular endothelial cells was studied by optical sectioning using a 3-D deconvolution microscopy system. Our results show that once bovine aortic endothelial cells (BAECs) reach confluency, the non-erythroidal spectrin is localized in the submembrane regions of the cells. Analysis of the intensity profiles of the non-erythroidal spectrin under isotonic and hypotonic conditions show that: (a) the width of the submembrane spectrin staining increases gradually with time within the first 5 minutes after the osmotic shock; (b) significant recovery is observed after 10 minutes even if the cells are maintained in hypotonic medium, and (c) spectrin distribution is altered by disrupting F-actin with latrunculin A but not by stabilizing F-actin with jasplakinolide. We suggest that cell swelling results in partial translocation of the submembrane spectrin to the cytosol and that it may play a major role in initiation of swelling-induced cellular events.     

Facilitated protoplasting in certain auxotrophic mutants of Corynebacterium glutamicum

Summary Auxotrophic mutants of Corynebacterium glutamicum strain ATCC 13 059 showed considerable variation in their ability to form protoplasts when treated with lysozyme and ethylene-diaminetetraacetic acid (EDTA) following growth in nutrient medium containing up to 2% glycine. The proportion of protoplasts formed by the parent strain was normally 10 to 15% whereas certain amino-acid auxotrophs formed protoplasts at much higher frequencies (>95%). There was no obvious correlation between the presence of specific auxotrophic markers or the number of rounds of mutagenesis and protoplasting ability. Strains which were most readily protoplasted were morphologically distinct from other auxotrophs and the parent strain but were similar to the parent strain in their sensitivity to lysozyme. However, isoleucine auxotrophs were more sensitive to penicillin G. All strains produced osmotically sensitive cells (lysed by dilution in water) after growth in glycine-containing media and lysozyme-EDTA treatment. These damaged cells could be distinguished from true protoplasts by their ability to recover on osmotically non-protective media if diluted in high osmotic pressure buffers. Protoplasts were regenerated on an osmotically protective medium (ET) in 48 to 72 h, at frequencies averaging 50–60%.     

Isolation of Staphylococcus aureus protoplasts using lysoamidase

The effect of the bacteriolytic enzyme preparation, lysoamidase, on Staphylococcus aureus 209P cells was studied. The protoplast formation was examined by spectrophotometric, biochemical and electron microscopic methods. Optimal conditions for isolation of S. aureus protoplasts were chosen. The susceptibility of S. aureus cells to lysoamidase depended on the culture age: the maximum effect was observed in the logarithmic growth phase. The protoplast yield was 80% when 1 M sucrose was used as an osmotic stabilizer. Lysoamidase caused local disruptures of the staphylococcus cell walls, which resulted in the formation of osmotically fragile spheroplasts and the release of protoplasts into the medium. The protoplasts obtained could retain 85-90% of the respiration activity and were able of cell wall regeneration.     

Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells

Summary When MDCK cells are cultured in MEM, they maintain a high concentration of three amino acids: glutamate (25mm), taurine (19 mm) and glycine (9 mm). With incubation of the cells in hypotonic media, the contents of these amino acids measured by HPLC are reduced in different time courses: taurine decreases most rapidly, followed by glutamate and glycine. All these losses are Na⁺ independent. To determine the transport mechanism activated by the hypotonic media, increasing external concentrations reaching 60 mm for nine different amino acids in Na⁺-free media were tested separately. For the five neutral (zwitterionic) amino acids, taurine, glycine, alanine, phenylalanine and tryptophan, cell contents increased linearly with external concentrations in hypotonic media, whereas in isotonic media only a slight rise was observed. The two anionic amino acids, glutamate and aspartate, were also increased linearly with their external concentrations in hypotonic media, but the changes were lower than those found for neutral amino acids. The presence of a negative membrane potential was responsible for this behavior since, using a K⁺ hypotonic medium which clamps the potential to zero, the glutamate content was found to increase linearly with an amplitude similar to the one observed for neutral amino acid. When external concentrations of two cationic amino acids, arginine and lysine, were increased in hypotonic media, only a small change, similar to that in isotonic media, was observed. These results indicate that a diffusion process for neutral and anionic amino acids is activated by a volume increase and it is suggested that an anion channel is involved.     

Ionic regulation of the haemolymph in the larvae of the dragonfly Aeshna cyanea (Müller) (Odonata, Anisoptera).

The ionic regulation of the haemolymph of larvae of Aeshna cyanea (Müller) was studied by means of two types of experiments. In the first the change in internal ionic composition was followed as a function of the time spent in a given experimental medium. These experiments led to the conclusion that: 1. the haemolymph composition does not change when larvae are starved in tap water for 10 days; 2. the haemolymph ionic concentrations (Na and Cl) have initial marked increase when the animals are kept in hypertonic media of diluted sea water; after 80 hours however, both concentrations stay constant. In a second series of experiments the internal ionic concentration was compared to a series of different concentrations of external media. From this, the relation between the internal and external ionic concentration was elaborated : in hypotonic media the internal Na and Cl concentrations stay constant, in hypertonic media there is a parallelism between the increase of the external concentration and haemolymph concentration, the internal Na concentration being always slightly hypertonic, the Cl concentration markedly hypotonic. Finally, when larvae are placed in glass distilled water, the internal Na and Cl concentrations begin to decrease after 60 hours.     

Sensitivity of the proliferation of normal and tumor cells to cytochalasin D

The effect of cytochalasin D, which is known to disrupt specifically actin cytoskeleton, on DNA replication was studied. The incubation of cultured mouse embryonic fibroblasts (MEF), cells of Balb/3T3 line and cells of minimally transformed clones 12 MC and 6 st/T CAK-7 line with cytochalasin D leads to inhibition of DNA synthesis. A complete inhibition of labeled index in MEF culture was observed after an 8 day incubation in cytochalasin D. Part of cells of clones 12 MC and 6 st/T were insensitive to cytochalasin D and continued to enter to S-phase even after a 10 day incubation. The transfer of cells into a fresh medium leads to a rapid restoration of DNA synthesis. Strongly transformed L cells were almost insensitive to cytochalasin D. Thus, the reorganization of actin cytoskeleton caused by cytochalasin D can inhibit the cycle of normal and minimally transformed cells. In the course of neoplastic progression, in the transformed cells there is a loss of dependence of cell proliferation on microfilament system.     

The relationship between the disassembly of microtubules during G1 and the enhancement of DNA synthesis by colchicine in mouse fibroblasts stimulated with peptide growth hormones

By immunofluorescent staining to visualize the cytoplasmic microtubular cytoskeleton in mouse fibroblasts we have ascertained that after a relatively short exposure of cells to colchicine, microtubules remain disassembled for a prolonged period of time after cells are transferred to a colchicine-free medium. In contrast to the persisting effects of colchicine, a brief exposure of cells to nocodazole first induces the expected disruption of microtubules followed by regeneration of the cytoskeleton within a few hours after removal of extracellular drug. These results shed light on our previous finding that quiescent mouse fibroblasts first treated with colchicine and then transferred to colchicine-free medium exhibit an enhanced proliferative response to EGF and insulin, whereas cells treated in a similar manner with nocodazole show no enhancement of DNA synthesis stimulated by peptide growth hormones. We conclude that cytoplasmic microtubules must remain disaggregated during the prereplicative G1 period in order for cells to exhibit the enhancing effects of the microtubule-disrupting drugs on DNA synthesis.