Effect of lipids on insect cell growth and expression of recombinant proteins in serum-free medium

The lipid emulsion components of a serum-free insect cell medium were varied and evaluated for effects on cell growth and recombinant protein expression. The growth of High-Five^TM cells was significantly affected by polyol Pluronic F-68 and Tween-80, but not by lipids. Pluronic was essential for cell growth, while Tween-80 was required to achieve maximum cell densities. A dose response effect was observed for Tween-80 with optimal cell growth at a concentration of 25 mg/l. Cholesterol had a minor effect on cell growth, but was essential for the expression of recombinant proteins. The expression of -galactosidase (-gal) was directly affected by cholesterol with optimal expression at a concentration of 5.4 mg/l. Vitamin E, important as an antioxidant to stabilize lipids, did not directly affect recombinant protein expression. Although lipids were not required for cell growth, the presence of lipids were required during the cell growth phase in order to achieve efficient infection with baculovirus. These studies help to define the important components, and range of concentrations, for lipid emulsions which can effectively replace serum in insect cell culture.Abbreviations -gal galactosidase (E.C. 3.2.1.23) - Sf-9 Spodoptera frugiperda - High-5 Trichoplusia ni 5Bl-4     

Effect of lipids on avian satellite cell proliferation, differentiation and heparan sulfate proteoglycan expression

The objective of this study was to determine the effects of fatty acids on the proliferation, differentiation, and expression of syndecan-4 and glypican-1 in avian myogenic satellite cells (SC). SC derived from the pectoralis major (PM) and biceps femoris (BF) muscles of the turkey and chicken were individually administered 8 different fatty acids in defined medium during proliferation. A parallel set of turkey SC was induced to differentiate. Highest levels of proliferation of turkey PM and BF SC occurred in cultures containing oleate. Linoleate and oleate were equipotent in supporting proliferation of chicken SC. Microscopic examination revealed that inclusion of docosahexaenoate or eicosapentaenoate was toxic towards both PM and BF SC from both species. Linolenate and arachidonate diminished levels of differentiation. Expression of glypican-1 varied between treatments to a greater extent with turkey BF than with PM SC. Expression in chicken PM and BF SC demonstrated a similar pattern in response to treatments. Turkey PM syndecan-4 expression varied between treatments, whereas expression in turkey BF SC was similar between treatments. Expression in chicken SC varied little between treatments. The results demonstrate species and muscle-specific differences in the parameters examined. It is proposed that changes in lipid raft receptor interactions may contribute to these observed differences.     

Effect of diethylstilboestrol on adipose-tissue lipids

1. The effect of diethylstilboestrol on the fatty acid composition of adipose-tissue lipids of the ox (Bos taurus) was studied. 2. The capsula adiposa (perirenal) was shown to contain more total saturated fatty acids, whereas more total unsaturated fatty acids were found in the panniculus adiposus (subcutaneous). 3. Significantly more stearic acid and linolenic acid were obtained from the capsula adiposa, whereas the panniculus adiposus contained more myristoleic acid, palmitoleic acid and oleic acid. 4. Implanting diethylstilboestrol significantly increased the deposition of the saturated fatty acids, particularly stearic acid. 5. A decrease in the deposition of total unsaturated fatty acids, myristoleic acid, palmitoleic acid and linoleic acid can also be attributed to the diethylstilboestrol treatment.     

Studies on cell transformation.

Seven different transformation stigmata of the transformed CHO cell line, including morphological characteristics, growth behavior, cell membrane biochemical properties, and failure of fibronectin deposition, are reversed by addition of cAMP derivatives to the medium. Simultaneously the microtubular pattern changes from a sparse, relatively random set to an orderly arrangement of tubules largely parallel to each other and to the long axis of the resulting fibroblastic cell. Agents like colcemid and cytochalasin B, respectively disorganizing microtubular and particular microfilamentous structures, prevent at least certain aspects of the reverse transformation reaction induced by cAMP in interphase cells. It is proposed that malignant transformation can be effected by damage to the microtubular and microfilamentous structures which changes cell constitution and behavior in two ways: (1) chromosomal instability is introduced which promotes continuous selection for variants better able to resist environmental signals to limit reproduction and (2) a variety of metabolic defects in biochemical processes such as specific membrane functions are introduced which may alter the growth responses of the cell. This picture offers a reasonable explanation for a number of aspects of normal and malignant cell behavior.     

Effect of stress on the composition of yeast lipids

Pigmented (Rhodotorula glutinis) and nonpigmented (Lipomyces starkeyi) yeasts were studied. Exogenous stressors (UV irradiation and methylene blue) were shown to change the composition of yeast lipids (especially the ratio of unsaturated fatty acids) and to increase the content of lipid peroxidation products formed (particularly in nonpigmented yeasts). In carotene-synthesizing yeasts, these stressors decreased the amount of carotenoids produced and did not affect the ratio between carotenoid pigments (beta-carotene, torulene, and torularhodin).     

Effect of cell substratum on lateral mobility of lipids in the plasma membrane of vascular endothelial cells

Bovine vascular endothelial cells can be maintained in a highly differentiated state in vitro, either by the addition of fibroblast growth factor (FGF) to the culture medium or by plating the cells on extracellular matrix (ECM)-coated dishes. Under these conditions the cells proliferate actively and at confluence form a tightly packed monolayer composed of nonoverlapping polarized cells. A fluorescence recovery after photobleaching method was used to determine the lateral mobility coefficient D of the lipophilic fluorescent probe, 5N-(hexadecanoyl)-aminofluorescein (HEDAF), in the basal and apical plasma membranes of endothelial cells under various culture conditions (cells on glass coverslips in the presence or absence of FGF, or cells plated on ECM in the exponential growth phase or at confluence). A heterogeneous distribution of lateral diffusion coefficients D was found in a given cell population. Nevertheless, for the basal membrane, a "mean" D value close to 2.0 x 10(-9) cm2/s was found for all the culture conditions. The "mean" D value of HEDAF in the apical pole was slightly higher when sparse cells were exposed to FGF (D = 2.2 x 10(-9) cm2/s) and was further enhanced when cells were growing or confluent on ECM-coated coverslips (D = 2.7 x 10(-9) cm2/s). On the other hand, when the cells were maintained in the absence of FGF on glass coverslips, similar "mean" D values were found in both cell poles (D = 2.0 x 10(-9) cm2/s). These results show that lateral mobility of lipids in endothelial plasmalemma varies in response to external factors such as FGF and the ECM.     

Effect of splenectomy on the expression of regulatory T cell activity.

The effect of adult splenectomy on the expression of suppressor and amplifier T cell activity was examined with respect to the serum antibody response to Type III pneumococcal polysaccharide (SSS-III) by using a sensitive radioimmunoassay. Suppressor T cell activity, as measured by the degree of low-dose paralysis induced, was not impaired in the least by splenectomy; however, amplifier T cell activity was almost completely eliminated within 7 days after splenectomy. These findings indicate that suppressor T cell activity is not confined solely to the spleen, the major site of antibody synthesis after immunization with SSS-III, and that the spleen may be an important site for the generation and/or maintenance of amplifier T cell activity.     

Effect of alveolar epithelial cell plasticity on the regulation of GM-CSF expression

Local pulmonary expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically important for defense of the pulmonary alveolar space. It is required for surfactant homeostasis and pulmonary innate immune responses and is protective against lung injury and aberrant repair. Alveolar epithelial cells (AEC) are a major source of GM-CSF; however, the control of homeostatic expression of GM-CSF is incompletely characterized. Increasing evidence suggests considerable plasticity of expression of AEC phenotypic characteristics. We tested the hypothesis that this plasticity extends to regulation of expression of GM-CSF using 1) MLE-12 cells (a commonly used murine cell line expressing some features of normal type II AEC, 2) primary murine AEC incubated under standard conditions [resulting in rapid spreading and loss of surfactant protein C (SP-C) expression with induction of the putative type I cell marker (T1α)], or 3) primary murine AEC on a hyaluronic acid/collagen matrix in defined medium, resulting in preservation of SP-C expression. AEC in standard cultures constitutively express abundant GM-CSF, with further induction in response to IL-1β but little response to TNF-α. In contrast, primary cells cultured to preserve SP-C expression and MLE-12 cells both express little GM-CSF constitutively, with significant induction in response to TNF-α and limited response to IL-1β. We conclude that constitutive and cytokine-induced expression of GM-CSF by AEC varies in concert with other cellular phenotypic characteristics. These changes may have important implications both for the maintenance of normal pulmonary homeostasis and for the process of repair following lung injury.     

Corticosteroid effect on Caco-2 cell lipids depends on cell differentiation

Previous studies from our laboratory have indicated that secondary hyperaldosteronism affects phospholipids of rat colonic enterocytes. To assess whether this represents a direct effect of mineralocorticoids on enterocytes, the role of aldosterone and dexamethasone in the regulation of lipid metabolism was examined in Caco-2 cells during development of their enterocyte phenotype. Differentiation of Caco-2 cells was associated with increased levels of triglycerides (TG) and cholesteryl esters (CE), a decreased content of cholesterol and phospholipids and changes in individual phospholipid classes. The phospholipids of differentiated cells had a higher content of n-6 polyunsaturated fatty acids (PUFA) and lower amounts of monounsaturated (MUFA) and saturated fatty acids than subconfluent undifferentiated cells. Differentiated cells exhibited a higher ability to incorporate [3H]arachidonic acid (AA) into cellular phospholipids and a lower ability for incorporation into TG and CE. Incubation of subconfluent undifferentiated cells with aldosterone or dexamethasone was without effect on the content of lipids, their fatty acids and [3H]AA incorporation. In contrast, aldosterone treatment of differentiated cells diminished the content of TG, increased the content of phospholipids and modulated their fatty acid composition. The percentage of n-6 and n-3 PUFA in phospholipids was increased and that of MUFA decreased, whereas no changes in TG were observed. The incorporation of [3H]AA into phospholipids was increased and into TG decreased and these changes were blocked by spironolactone. Treatment of differentiated cells with dexamethasone increased their CE content but no effect was identified upon other lipids, their fatty acid composition and on the incorporation of [3H]AA. As expected for the involvement of corticosteroid hormones the mineralocorticoid and glucocorticoid receptors were identified in Caco-2 cells by RT-PCR. The results suggest that aldosterone had a profound influence on lipid metabolism in enterocytes and that its effect depends on the stage of differentiation. The aldosterone-dependent changes occurring in phospholipids and their fatty acid composition may reflect a physiologically important phenomenon with long-term consequences for membrane structure and function.     

Effect of platelet-activating factor receptor expression on CHO cell motility

Tumor cell migration may favor local mass expansion and metastasis dissemination. Several tumors were found to express the receptor for platelet-activating factor (PAF), a potent mediator of leukocyte chemotaxis and endothelial cell migration. However, its functional role on tumor cells is largely unexplored. In the present study, we evaluated the motogenic effect of PAF on Chinese hamster ovarian (CHO) cancer cells transfected with the human PAF-receptor cDNA (CHO PAF-R). By using time-lapse recording, we detected a rapid motogenic response to PAF stimulation on CHO PAF-R, whereas no effect was evident on vector-only transfected cells. Such an effect was observed on scattered cell motility, on cells seeded on a fibronectin- or collagen-coated surface, and on migration of confluent monolayer cells. Cell speed increased at 1 h and was maximal 6-8 h after PAF stimulation on CHO PAF-R. Concomitantly, PAF induced marked changes in cytoskeleton actin distribution with cell contraction, assembling of stress fibers, and polar foci of adhesion. In conclusion, the present study demonstrates that PAF is a potent inducer of tumor cell motility, thus suggesting a role for this mediator in tumor growth and dissemination.     

Effect of aminazin on the enzymatic oxidation of lipids]

An inhibitory effect of chlorpromazine on the enzymatic NADPH-dependent lipid peroxidation in rat liver microsomal fraction was found. This inhibition was caused by the 1) antioxidative effect of hydroxy-derivatives appearing during the oxidative metabolism of chlorpromazine with NADPH-dependent microsomal oxygenases, and by the 2) competition for reduced components of electron-carriers between the NADPH-dependent processes: chlorpromazine metabolism and lipids peroxidation.     

Effect of chlorophenols on the membrane lipids of bacterial cells

Chlorophenols, widespread soil and water contaminants and often degradation products of some pesticides, are a potential stress factor for survival of environmental bacteria. The effect of pentachlorophenol (PCP) and 2,4-chlorophenol (2,4-CP) on the growth, amount of lipid, and fatty acid composition in the membrane lipids was examined in a strain of the bacterium Kocuria varians, able to degrade chlorophenols. The index of fatty acid unsaturation in two main membrane lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased in the presence of chlorophenols. Transformation of stearic acid into oleic acid was significantly increased by PCP addition only in PE, but conversion of oleic acid into linoleic acid was blocked by PCP and 2,4-CP in both PC and PE. This observation may indicate that while Δ⁹ desaturase was sensitive mainly to 2,4-CP, activity of Δ¹² desaturase was inhibited by both PCP and 2,4-CP.