Buoyant density constancy during the cell cycle of Escherichia coli

Cell buoyant densities were determined in exponentially growing cultures of Escherichia coli B/r NC32 and E. coli K-12 PAT84 by equilibrium centrifugation in Percoll gradients. Distributions within density bands were measured as viable cells or total numbers of cells. At all growth rates, buoyant densities had narrow normal distributions with essentially the same value for the coefficient of variation, 0.15%. When the density distributions were determined in Ficoll gradients, they were more than twice as broad, but this increased variability was associated with the binding of Ficoll to the bacteria. Mean cell volumes and cell lengths were independent of cell densities in Percoll bands, within experimental errors, both in slowly and in rapidly growing cultures. Buoyant densities of cells separated by size, and therefore by age, in sucrose gradients also were observed to be independent of age. The results make unlikely any stepwise change in mean buoyant density of 0.1% or more during the cycle. These results also make it unlikely that signaling functions for cell division or for other cell cycle events are provided by density variations.     

Selective loss of chemokine receptor expression on leukocytes after cell isolation

Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB) plus erythrocyte lysis and density gradient isolation (Ficoll). Most WB monocytes were CCR2+ (93.5 ± 2.9%) whereas 32.8 ± 6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001). Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4 ± 7.5% and 57.1 ± 5.5%; Ficoll: 29.5 ± 2.2% and 5.4 ± 4.3% respectively) (p<0.01). Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5 ± 0.4 and Ficoll: MFI 3.3 ± 0.1) (p<0.05). Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2 ± 4.5% and Ficoll: 55 ± 4.1%) (p<0.01). The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured.     

High carbohydrate fat-free diet modulates epitope expression of LDL-apoB-100 and interaction of LDL with human fibroblasts

High carbohydrate diets are known to increase the concentration of very low density lipoprotein (VLDL) and to lower the concentrations of low density lipoprotein (LDL) and high density lipoprotein (HDL) in plasma. Such diets also alter lipoprotein compositions and metabolism. The aims of the present study were to assess in detail the effects of a virtually fat-free high carbohydrate (CHO) diet (CHO greater than 85% and fat less than 1% of calories) on various aspects of LDL. Thirteen healthy normolipidemic volunteers ate a basal "American" diet and the CHO diet for 7 days each in a forward or reverse sequence. Fasting blood samples were drawn at the ends of each study period and analyzed for lipoprotein lipid and apolipoprotein concentrations. Compositions of LDL particles isolated by ultracentrifugation were characterized chemically, LDL sizes were assessed by nondenaturing gradient electrophoresis on 2-16% gels, and association and degradation of LDL with normal human skin fibroblasts were quantified in cell cultures. Immunoreactivities of apoB in LDL were tested in solid phase competitive binding radioimmunoassays using five monoclonal anti-LDL antibodies that reacted with defined epitopes of apoB-100. The study diet produced consistent decreases of LDL cholesterol and apoB concentrations by 25% and 17%, respectively. LDL compositions were altered. Mean LDL triglycerides increased 3% to 4% of total LDL mass (P less than 0.004), and LDL particle sizes decreased (P less than 0.01). In radioimmunoassays that contained monoclonal antibody B1B3, an antibody that inhibits binding of LDL to the LDL receptor, the mean ED50 value for LDL protein was reduced from 3.75 to 2.66 micrograms (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-independent growth of human ovarian carcinoma cells

Evidence in the literature suggests that cancer cell growth in vitro is generally not sensitive to external calcium. A human ovarian carcinoma cell line (SKOV3) retained 60% of its normal growth in Dulbecco modified Eagle's medium (DME) when the calcium concentration was reduced from 3 mM to 10 microM. Chinese hamster ovary cells (CHO) were growth-arrested in media containing less than 500 microM calcium. In low-calcium (10 microM) DME, 10 microM of a calmodulin antagonist W7 inhibited the growth of SKOV3 cells by more than 90%, while 100 microM of its inactive analog W5 was mildly inhibitory (20%). The growth inhibition by W7 was antagonized by increasing calcium concentrations in the culture media, while the inhibition by W5 was calcium-independent. The phorbol ester TPA was also effective in antagonizing W7's growth inhibition in low-calcium DME, suggesting that the W7 effect is mediated via protein kinase C inhibition. SKOV3 total cellular protein kinase C activity was 1.6 times higher than CHO cells when incubated in normal DME. When incubated in low-calcium DME, a large drop in protein kinase C activity in the CHO cells was observed while the enzyme activity was unchanged in the SKOV3 cells. Our data suggest that these human ovarian tumor cells have altered cellular calcium regulatory processes associated with the defective down-regulation of protein kinase C. This defect may confer these cells the ability to proliferate independently of the external calcium concentration. Targeting the cellular signal transduction components may be useful in cancer chemotherapy.     

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.     

Effect of the growth state on protein turnover in two lines of cultured BHK cells

The adherence, phagocytic activity and buoyant density of mouse peritoneal exudate colony forming units (CFU-PE) were investigated. There was a significant enrichment in the proportion of CFU-PE in the adherent cells population, defined as cells adhering to a plastic surface within 30 minutes of incubation. The phagocytic activity of CFU-PE was studied by incubating exudate cells with iron particles for 45 minutes. The cells were then separated into phagocytic and non-phagocytic cell fractions by passing the incubation mixture through a magnetic field. A significant enrichment of CFU-PE was seen in the phagocytic cell fraction. When exudate cells were fractionated in a Ficoll discontinuous density gradient, more than 88% of CFU-PE were recovered at the 16/18% and 18/20% interfaces. It is concluded that CFU-PE are adherent cells, have strong phagocytic activity and have a buoyant density between 1.0562 and 1.0703. When bone marrow cells were studied by these techniques, the committed stem cells for both granulocytes and macrophages (CFU-C) were enriched in both non-adherent cell and non-phagocytic cells populations. In the Ficoll density gradient, CFU-C banded at a heavier density region than CFU-PE.     

Tetrahydrofolate increases suspension growth of dihydrofolate reductase‐deficient chinese hamster ovary DG44 cells in chemically defined media

Adaptation of dihydrofolate reductase (DHFR)‐deficient Chinese hamster ovary (CHO) DG44 cells to chemically defined suspension culture conditions is a time‐consuming and labor‐intensive process because nonadapted DHFR‐deficient CHO DG44 cells normally show poor growth in chemically defined medium (CDM). We examined the effects of folate derivatives, ribonucleotides, and nucleobases on the growth of suspension‐adapted DHFR‐deficient CHO DG44 cells in CDM. Among the tested additives, tetrahydrofolate (THF) was identified as an effective component for increasing cell growth. THF supplementation in the range of 0.2–359 μM enhanced cell growth in in‐house CDM. Addition of 3.6 μM THF to in‐house CDM resulted in a more than 2.5‐fold increase in maximum viable cell density. Moreover, supplementation of six different commercial CDMs with 3.6 μM THF yielded up to 2.9‐fold enhancement of maximum viable cell density. An anchorage‐ and serum‐dependent DHFR‐deficient CHO DG44 cell line was adapted within two consecutive passages to suspension growth in in‐house CDM supplemented with 3.6 μM THF. These data indicate that supplementation of chemically defined cell culture media with greater than 0.2 μM THF can help achieve a high density of suspension‐adapted DHFR‐deficient CHO DG44 cells and may facilitate rapid adaptation of nonadapted DHFR‐deficient CHO DG44 cells to suspension culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1539–1546, 2016     

A simple method for reconstitution of CHO cell and human fibroblast acyl coenzyme A: cholesterol acyltransferase activity into liposomes

A new method for reconstituting acyl coenzyme A: cholesterol acyltransferase (ACAT) activity from either Chinese hamster ovary (CHO) or human fibroblast cell extracts into cholesterol-phosphatidylcholine liposomes is described. The method is rapid (less than 60 min) and easy to perform. The procedure involves solubilizing the cell extracts with deoxycholate followed by dilution into preformed liposomes. Ficoll gradient analysis demonstrated that, after reconstitution, almost all of the detectable ACAT activity co-migrated with the liposomes. Exogenous cholesterol in the liposomes was absolutely necessary for providing ACAT activity, but not for incorporation of the ACAT enzyme into the vesicle bilayer. Human fibroblast cell extracts prepared from cells grown in medium containing 10% fetal calf serum were found to contain a 10-fold higher microsomal ACAT activity compared to extracts from cells grown in 10% delipidated fetal calf serum. In contrast, when the ACAT activity from these extracts was measured using the reconstitution assay, there was no difference in the specific activities. These results support our previous work (Doolittle, G. M., and T. Y. Chang. 1982. Biochim. Biophys. Acta. 713: 529-537; and Chang, C. C. Y., et al. 1986. Biochemistry. 25: 1693-1699), and suggest that cholesterol regulates ACAT activity in CHO cells and human fibroblasts by mechanism(s) other than modulation of the amount of enzyme.     

Elevated levels of the alpha 5 beta 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells

We report here on gene transfer studies designed to investigate the function of the alpha 5 beta 1 integrin and its role in transformation. Transfection of the human alpha 5 and beta 1 cDNAs into transformed Chinese hamster ovary (CHO) cells followed by methotrexate-induced amplification yielded clonal cell lines overexpression this fibronectin receptor. The overexpressors deposited more fibronectin in their extracellular matrix and migrated less than control cells. In addition, they showed reduced saturation density and reduced ability to grow in soft agar. The overexpressor cells, unlike the control CHO cells, were nontumorigenic when injected subcutaneously into nude mice. The results indicate that extracellular matrix recognition by the alpha 5 beta 1 integrin plays a role in the control of cell proliferation and suggest that a reduction of this fibronectin receptor may be responsible for the acquisition of anchorage independence by transformed cells.     

微囊化重组CHO细胞制备和培养条件的优化

微囊化重组基因细胞移植治疗肿瘤是一种新兴的肿瘤基因治疗方法,然而由于目前微囊化细胞规模化制备和培养技术还不成熟,阻碍了其在临床治疗中的推广与应用。以重组CHO细胞为模型,考察了不同的微囊制备和培养条件对微囊化细胞生长和内皮抑素表达的影响。实验表明,种子细胞所处的生长阶段和细胞接种密度对微囊化细胞生长和内皮抑素表达的影响较大,对数生长期的细胞进行包囊并且细胞接种密度为1×106~2×106cells/mL微囊时微囊内细胞生长良好、内皮抑素表达量高。微囊制备时间对细胞活性和内皮抑素表达也有较大的影响,制备时间延长对细胞的损伤增大,因此制备时间应控制在5h以内。生物微胶囊在制备过程中会造成细胞损伤,而体外培养是恢复细胞活性的良好方法,在培养过程中微囊接种量为5%时对细胞生长和内皮抑素表达有利。     

Isolation of mastocytes from rat peritoneal fluid using continuous Ficoll gradients

A method for mast cells purification from rat peritoneal fluid is described. The method consists of a continuous Ficoll gradient between 20 and 22,5% (w/v) Ficoll and the cells are obtained with an 88% retrieval. Purity and viability were of 95% and 97% respectively. The cells so purified were functional against the compound 48/80 and the spontaneous secretion value was less than 8%.     

G2 cell cycle arrest induced by glycopeptides isolated from the bovine cerebral cortex

The ability of glycopeptides, isolated from bovine cerebral cortex, to alter cell division was studied by cell-cycle analyses. The results showed that glycopeptides arrested baby hamster kidney (BHK)-21 cells and Chinese hamster ovary (CHO) cells in the G2 phase of the cell cycle. Upon removal of the growth inhibition from arrested BHK-21 cells, the mitotic index in colchicine-treated cultures increased from 5 to 40% within 6 h and the increase in mitotic activity was accompanied by a complete doubling of all arrested cells within this 6- h time period. Determination of DNA content in growth-arrested BHK-21 cells showed that growth-arrested cells contained about twice the DNA of control cell cultures. Although CHO cells treated in a like manner with growth inhibitor could not be arrested for the same length of time as BHK-21 cells (18 h vs. 72 h before initiation of escape) and to the same degree (60% of the cell population vs. 99% of BHK-21 cells), the escape kinetics of CHO cells did indicate a G2 arrest. Approximately 3.5 h after escape began, CHO cell numbers in treated cultures attained the cell numbers found in control cultures. This rapid growth phase occurring in less than 4 h indicated that the growth inhibitor induced a G2 arrest-point in CHO cells that was not lethal since the entire arrested cell population divided.     

Effect of iron on adherence and cytotoxicity of Entamoeba histolytica to CHO cell monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.     

Engineering Chinese hamster ovary (CHO) cells to achieve an inverse growth – associated production of a foreign protein, β-galactosidase

Protein synthesis in mammalian cells can be observed in two strikingly different patterns: 1) production of monoclonal antibodies in hybridoma cultures is typically inverse growth associated and 2) production of most therapeutic glycoproteins in recombinant mammalian cell cultures is found to be growth associated. Production of monoclonal antibodies has been easily maximized by culturing hybridoma cells at very low growth rates in high cell density fed- batch or perfusion bioreactors. Applying the same bioreactor techniques to recombinant mammalian cell cultures results in drastically reduced production rates due to their growth associated production kinetics. Optimization of such growth associated production requires high cell growth conditions, such as in repeated batch cultures or chemostat cultures with attendant excess biomass synthesis. Our recent research has demonstrated that this growth associated production in recombinant Chinese hamster ovary (CHO) cells is related to the S (DNA synthesis)-phase specific production due to the SV40 early promoter commonly used for driving the foreign gene expression. Using the stably transfected CHO cell lines synthesizing an intracellular reporter protein under the control of SV40 early promoter, we have recently demonstrated in batch and continuous cultures that the product synthesis is growth associated. We have now replaced this S-phase specific promoter in new expression vectors with the adenovirus major late promoter which was found to be active primarily in the G1-phase and is expected to yield the desirable inverse growth associated production behavior. Our results in repeated batch cultures show that the protein synthesis kinetics in this resulting CHO cell line is indeed inverse growth associated. Results from continuous and high cell density perfusion culture experiments also indicate a strong inverse growth associated protein synthesis. The bioreactor optimization with this desirable inverse growth associated production behavior would be much simpler than bioreactor operation for cells with growth associated production. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of cycloheximide on development of methotrexate resistance of Chinese hamster ovary cells treated with inhibitors of DNA synthesis.

We examined the effects of 18 h of incubation of Chinese hamster ovary (CHO K1) cells with cycloheximide, hydroxyurea, and aphidicolin. Treatment of cells with cycloheximide alone at a concentration adequate to inhibit DNA synthesis to less than 10% of control was significantly less cytotoxic and clastogenic than treatment with hydroxyurea or aphidicolin, did not induce unbalanced cellular growth, and had no effect on the frequency of resistant cells in methotrexate selections compared with control cells. When combined with hydroxyurea or aphidicolin and compared with the effects of either drug alone, cycloheximide blocked the induction of unbalanced growth during drug treatment, reduced the frequency of chromosomal aberrations in recovering cell populations, and decreased cell killing. In addition, the increased frequency of methotrexate-resistant cells observed after treatment with hydroxyurea or aphidicolin was eliminated when cycloheximide was present during drug treatment.     

Structure of the fetal sheep brain in experimental growth retardation

A quantitative morphometric study of brain development has been made in growth-retarded fetal sheep. Intrauterine growth retardation was induced by removal of endometrial caruncles in the ewe prior to conception thereby reducing the size of the placenta in a subsequent pregnancy. Total brain and cerebellar weights were reduced by 21% (P less than 0.002) and the cerebrum by 20% (P less than 0.05) in the growth-retarded fetuses at 139 +/- 1 day (term = 146 days) compared with age matched control fetuses. Measurements of mean neuronal diameters were made on Purkinje cells, cerebellar granule cells, cortical cells in the motor and visual areas and hippocampal pyramidal cells; none were significantly different from control values. In growth-retarded fetuses compared with controls, there was a significant reduction in the thickness of the motor and visual cortices and the numerical density of neurones was significantly higher in these areas. In the cerebellar vermis, the number of Purkinje cells per unit surface area of Purkinje cell layer was higher, the numerical density of granule cells was significantly higher concomitant with a reduction in the area of the inner granular layer, and the area of the molecular layer was also reduced. In the hippocampal formation, the numerical density of pyramidal neurones was higher and the width of the stratum moleculare (dentate gyrus) was reduced. Migration of pyramidal neurones from the germinal layer to stratum pyramidale was not affected. These findings indicate that intrauterine growth retardation does not markedly affect cell size or neuronal migration (in the hippocampus) but does cause a significant reduction in the growth of the neuropil in the cerebellum, motor and visual cortices and the hippocampal formation.     

Induction of anthocyanin synthesis in relation to embryogenesis in a carrot suspension culture: Correlation of metabolic differentiation with morphological differentiation

A system in which anthocyanin synthesis could be induced under a defined condition, was established in a carrot suspension culture. A cell suspension culture of carrot ( Daucus carota L. cv. Kurodagosun) was subcultured for more than a year in a medium containing 5 × 10⁻⁷ M 2,4-dichlorophenoxyacetic acid (2,4-D). At every subculture the cultures were sieved through nylon screens and the cells and cell clusters collected in the size range of 31–81 μm were transferred to a fresh medium. When the cells were transferred to a medium without auxin, synthesis of anthocyanin was induced. Zeatin promoted anthocyanin synthesis in a medium lacking auxin, with maximum yields of anthocyanin obtained at 10⁻⁷ to 10⁻⁸ M zeatin, 2,4-D at higher concentrations than 10⁻⁷ M inhibited anthocyanin synthesis completely. The sieved cells were fractionated by Ficoll density gradient centrifugation. Somatic embryos were formed in the fraction of higher density (>14% of Ficoll) in a medium containing 10⁻⁷ M zeatin but lacking auxin, while synthesis of anthocyanin was hardly observed. On the other hand, cells in the fraction of lower density (<12% of Ficoll) synthesized anthocyanin in the same medium, but formed few embryos. Forty to fifty percent of the total cells in this lighter cell fraction synthesized anthocyanin at a maximum. The similarity between anthocyanin synthesis and embryogenesis was observed in the time course as well as in the effects of growth regulators. The correlation between metabolic and morphological differentiation is discussed.     

Effects of heat treatment and concentration of fish serum on cell growth in adhesion culture of Chinese hamster ovary cells

The effects of heat treatment and concentration of fish serum (FS) on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of recombinant Chinese hamster ovary (CHO) cells, DR1000L4N, were investigated. The addition of heat treated FS instead of non-heat-treated FS improved cell growth in terms of cell density, which reached 60% that in 10% fetal calf serum (FCS)-containing medium (FCS medium). A decrease in FS concentration from 10 to 1.25% markedly increased cell density, which was 79% that in 10% FCS medium. The combination of heat treatment at 56 °C and the addition of FS at a low concentration (1.25%) showed an additive effect on cell growth and resulted in the same cell density as that in 10% FCS medium, whereas the hGM-CSF concentration in the culture using FS-containing medium (FS medium) was approximately 50% that in 10% FCS medium. The total lipid concentration in FS was more than three fold that in FCS. The effect of decreasing FS concentration on cell growth may be due to the low lipid concentration in FS medium, because addition of the lipids extracted from FS to 10% FCS and 1.25% FS media markedly decreased cell density. Consequently, the addition of heat-treated FS at low concentrations to medium may be useful for the growth of CHO cells without FCS.     

Phosphatidylcholine deficiency upregulates enzymes of triacylglycerol metabolism in CHO cells

We studied the regulation of triacylglycerol (TAG) metabolism by phosphatidylcholine (PC) in CHO MT58 cells, which are deficient in PC synthesis because of a temperature-sensitive CTP:phosphocholine cytidylyltransferase. At the permissive growth temperature (34 degrees C), these cells contained 49% less TAG and 30% less PC than wild-type CHO K1 cells. Treatment with dipalmitoylphosphatidylcholine normalized both the PC and TAG levels. Despite low TAG levels, the incorporation of [14C]oleate into TAG was increased in CHO MT58 cells. The in vitro de novo synthesis of TAG and the activity of diacylglycerol acyltransferase were 90% and 34% higher, respectively. Two other key enzyme activities in TAG synthesis, acyl-CoA synthetase and mitochondrial glycerol-3-phosphate acyltransferase (GPAT), increased by 48% and 2-fold, respectively, and mitochondrial GPAT mRNA increased by approximately 4-fold. Additionally, TAG hydrolysis was accelerated in CHO MT58 cells, and in vitro lipolytic activity increased by 68%. These studies suggest that a homeostatic mechanism increases TAG synthesis and recycling in response to PC deficiency. TAG recycling produces diacylglycerol and fatty acids that can be substrates for de novo PC synthesis and for lysophosphatidylcholine (lysoPC) acylation. In CHO MT58 cells, in which de novo PC synthesis is blocked, lysoPC acylation with fatty acid originating from TAG may represent the main pathway for generating PC.