Increased protein synthetic capacity in vitro of rat liver rough endoplasmic reticulum following starvation

There is a dramatic rise in the protein synthetic activity of rough endoplasmic reticulum (RER) of liver from starved and starved-refed rats, compared to normal controls. This observation is similar to the doubling of protein synthetic activity of rat liver RER observed 20 hours after partial hepatectomy (1). Oxidised glutathione (GSSG), a potent inhibitor of protein synthesis by normal RER, was a much less effective inhibitor when assayed with RER from the livers of starved rats, this again is in parallel with our earlier observations in regenerating liver. This common pattern of response is discussed in terms of a cellular control system which responds to changes in the cytoplasmic sulphydryl concentration.     

Permeability of cultured megakaryocytopoietic cells of the rat to dimethyl sulfoxide

The permeability of the membrane of the rat megakaryocytopoietic cell to dimethyl sulfoxide was measured to assess its availability to the intracellular compartment. The method used was osmotic, and measured the initial loss of cell water followed by a reswelling to isotonic volume when cells were placed in culture media containing 0.6 M DMSO. Values for the hydraulic coefficient, Lp, the permeability of the membrane to DMSO, wRT , and the reflection coefficient were calculated from the equations of Kedem and Katchalsky . The average value at 25 degrees C for Lp was 0.46 micron min-1 atm1 ; wRT was 9.3 micron min-1, and the reflection coefficient was 0.65. At these cell volumes, 50% equilibration occurred in 5 sec. Cells equilibrated in 0.6 M DMSO increased their volume of osmotically inactive water. Coupled with this phenomenon of stabilization of water was a reduction in the hydraulic coefficient by 50%. These findings are discussed in the context of current hypotheses about cellular viability during freezing and thawing in the presence and absence of cryoprotectants.     

Enhanced elevations of hypo-osmotic shock-induced cytosolic and nucleic calcium concentrations in tobacco cells by pretreatment with dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) is a dipolar aprotic solvent widely used in biological assays. Here, we observed that DMSO enhanced the hypo-osmotically induced increases in the concentration of Ca²⁺ in cytosolic and nucleic compartments in the transgenic cell-lines of tobacco (BY-2) expressing aequorin.     

Lipid composition of Friend leukemia cells following induction of erythroid differentiation by dimethyl sulfoxide

The effects of dimethyl sulfoxide (DMSO)-induced differentiation of Friend leukemia cells in vitro on the lipid composition of these cells have been examined. DMSO had no early effect on the incorporation of either [¹⁴C] glycerol or [³H] methyl choline chloride into the total lipids or individual phospholipids of Friend cells up to 240 min after addition of the inducer. Examination of DMSO-diferentiated Friend cell phospholipids revealed a percentage composition which was similar to control cells, with phosphatidylcholine and phosphatidylethanolamine in both uninduced and differentiated cells accounting for over 75% of the total phospholipid. Sphingomyelin levels were significantly lower in Friend cells than in normal adult mouse erythrocytes, and differentiation of murine erythroleukemia cells resulted in a further lowering of this phospholipid. In contrast, a significant increase in the level of phosphatidylethanolamine occured as a result of maturation. Fatty acid analysis of major lipid classes of differentiated Friend cells showed significant reduction in saturation, but no alteration in chain length in comparison to undifferentiated cells. A pronounced decrease in the cellular content of both free and esterified cholesterol, which resulted in a 45% decrease in the ratio of cholesterol/phospholipids, occurred in cells differentiated by the polar solvent. The findings indicate that erythrodifferentiation induced by DMSO results in a variety of changes in the lipid composition of the membranes of Friend leukemia cells.     

Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration.

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.     

Countercurrent distribution in a 2-phase polymer system of cultured cells following their exposure to trypsin, versen, dimethyl sulfoxide and suboptimal temperature

A study was made of the effects of trypsin (0.25%) EDTA (0.02%), DMSO (10%) and suboptimal temperatures (16-18 degrees C) on the line L cells of monolayer and suspension cultures by the method of counter-current distribution in two-phase polymer system Dextran-500/poly (ethylene) glycol-6000. It has been shown that these agents cause the increase in number of cells with low partition coefficient, which is most expressed in the population of monolayer subline cells.     

Studies on the assembly and stability of the metal-thiolate clusters of metallothionein in dimethyl sulfoxide.

Solvent perturbation studies in 100% dimethyl sulfoxide (Me2SO) have been carried out on rabbit liver metallothionein (MT) in an effort to learn more about the factors stabilizing the three-dimensional structure and the mechanism of cluster formation. As indicated by the electronic absorption spectra of Co7-metallothionein, the reconstituted protein preserves its structural integrity in this solvent. Minor spectral differences between water and Me2SO were fully reversible. The titration of apoMT with cobalt(II) in Me2SO, followed by UV-visible-near-infrared electronic absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopy, indicate that the protein can refold in this solvent. A comparison with the previous titration data in water reveals that the first four titration steps in both solvents are identical, indicating a thermodynamically controlled folding process. However, the reversed order of the cluster completion between Me2SO and water may suggest the involvement of a kinetically controlled folding process in the last three titration steps. A new cluster form developed with approximately nine Co(II) equivalents.     

Effect of dimethyl sulfoxide on transformed rat Schwann cells

Cultured rat Schwann cells transformed by Simian Virus 40 (SV40) have previously been shown to retain their ability to synthesize myelin-associated galactosylceramide and sulfatide. Little is known about the mechanism regulating galactosphingolipid synthesis in Schwann cells. We have found that growing the transformed Schwann cells in the presence of dimethyl sulfoxide (DMSO) markedly inhibits the incorporation of [35S]sulfate into sulfatide, in a time- and dose-dependent manner. The concentration of DMSO which resulted in a half-maximal inhibition after 6 days of incubation was 0.5%, and the incubation time required for a half-maximal effect at 1.0% DMSO was approximately 4 days. In contrast, DMSC did not affect the incorporation of [35S]sulfate into glycosaminoglycans. In addition, DMSO treatment has little effect on the synthesis of cellular DNA, proteins and lipids. When transformed Schwann cells were treated with DMSO, a substantial decrease in the incorporation of [3H]galactose into galactosylceramide was observed. The concentration of DMSO which resulted in a half-maximal inhibition of galactosylceramide synthesis was approximately 0.5%, similar to the concentration required for a similar effect on sulfatide synthesis. However, the incubation time required for a half-maximal inhibitory effect on galactosylceramide synthesis at 1.0% DMSO was less than 1 day, which was substantially shorter than the time required for the inhibition of sulfatide synthesis at this concentration. This finding is consistent with the interpretation that treatment with DMSO inhibits the synthesis of galactosylceramide, a precursor of sulfatide, which results in a decrease in the synthesis of sulfatide during a prolonged incubation of DMSO.     

Alteration of the electrophoretic mobility of human peripheral blood mononuclear cells following treatment with dimethyl sulfoxide

Studies have been conducted to determine the effects of DMSO and freezing on the electrophoretic distribution of peripheral blood mononuclear cells. Sodium [51Cr]chromate was used to label the cells, and the distributions of cell number and cell-associated radioactivity were determined. Cells treated with DMSO had a narrower distribution of electrophoretic mobilities when compared with those not treated. DMSO-treated cells also demonstrated a more homogeneous distribution of radioactivity relative to the cell distribution than did the nontreated cells. The freezing of DMSO-treated cells did not result in any additional alteration of electrophoretic pattern compared to DMSO treatment alone. Analysis by linear categorization techniques indicated that the DMSO-treated and nontreated cells were completely distinguished by their electrophoretic behavior.     

Enhanced synthesis of type IV collagen in cultured arterial smooth muscle cells associated with phenotypic modulation by dimethyl sulfoxide

The effect of dimethyl sulfoxide (DMSO) on synthesis of basement membrane collagen in cultured smooth muscle cells was evaluated. DMSO promoted phenotypic modulation of cells from the synthetic state to the contractile state accompanied by formation of basement membranes. By immunofluorescence using monospecific antibody against type IV collagen, type IV collagen was identified not only in the cell cytoplasms but intensely along the cell surfaces in the cultures treated with DMSO for 7 days, as compared with untreated cultures. Electron microscopic immunohistochemistry revealed the presence of type IV collagen both in the basement membrane region and in the rough endoplasmic reticulum of DMSO-treated cells. Such an enhancement of type IV collagen synthesis appears to be expressed as a result of the phenotypic changes of smooth muscle cells to the contractile state modulated by DMSO.     

Aluminum neurotoxicity is reduced by dantrolene and dimethyl sulfoxide in cultured rat hippocampal neurons

Cell death was reduced in cultured rat hippocampal cells treated with aluminum chloride by dantrolene and dimethylsulfoxide, indicating aluminum toxicity may be mediated through release of calcium from intracellular stores and oxidative stress. Cell death was reduced to a lesser degree by cycloheximide and actinomycin D, indicating some evidence for apoptosis, however apoptosis did not appear to be a major cause of cell death from aluminum toxicity.     

Improvement of monoclonal antibody production in hybridoma cells by dimethyl sulfoxide

Hybridoma cultures are routinely used as a source for monoclonal antibody (mAb) production necessary for preclinical evaluation. However, these cultures typically have low volumetric and specific productivities. In this article, we examined the use and the timing of addition of dimethyl sulfoxide (DMSO) as a medium additive to improve mAb production in our hybridoma clone 19 (c19) cultures. From shake flask studies, we defined the optimal DMSO concentration and time of addition for improved productivity. This timing coordinated with high cell viability and density. Hybridoma cultures treated with DMSO up to 0.3% (v/v) possessed cell densities and viabilities comparable to untreated control. We demonstrated that 0.2% (v/v) DMSO added to shake flask cultures at their maximal viable cell densities resulted in a 2-fold increase in specific mAb production. This procedure was scaleable up to 20 L Cellbags (Wave Bioreactors) with similar titer improvement. Moreover, DMSO treatment did not affect the bioactivity or glycosylation profiles of the mAb.     

Optimal conditions for cryopreservation of primary chicken embryo kidney cells with dimethyl sulfoxide

Conditions were evaluated for optimum cryopreservation of primary chicken embryo kidney (CEK) cells. The recovery of viable CEK cells was best (50.8% viability) when the concentration of dimethyl sulfoxide (DMSO) in the freezing medium was 20% (v/v). The viability of primary CEK cells was not influenced by the concentration of calf serum in the freezing medium, the duration of storage at −70°C before storage in liquid nitrogen, cell concentration, or the method of addition or dilution of DMSO. Thawed cells recovered and grew in complete growth medium similarly to cells freshly isolated from kidney, and influenza viruses produced plaques in the monolayer. The cryopreservation procedures described here may facilitate maintenance of a standard stock of primary CEK cells for laboratories where preparation of primary CEK cells is not an option.     

Enhanced intracellular accumulation of recombinant HBsAg in CHO cells by dimethyl sulfoxide

The intracellular hepatitis B surface antigen (HBsAg) content per cell was significantly increased by 7.2-fold in the culture of recombinant CHO cells with 1.5% dimethyl sulfoxide (DMSO), while the HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. The significant accumulation of HBsAg within rCHO cells by DMSO stimulation was testified with flow cytometry measurements. Electron microscopy was applied to show that the dilated areas scattered over whole cytoplasm within rCHO cells in response to DMSO, and further revealed that intracellular HBsAg was localized to these areas with immunogold labeling. The failure of intracellular HBsAg virus-like particle assembly was revealed to be closely associated with the HBsAg accumulation within DMSO-stimulated rCHO cells on the basis of sucrose gradient analysis of cell extract. This work provided the details to further understand the HBsAg accumulation within rCHO cells in response to DMSO.