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1.
Regulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [14C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein-free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.  相似文献   

2.
E Fuchs  H Green 《Cell》1981,25(3):617-625
Vitamin A is known to exert an important influence on epithelial differentiation. The fetal calf serum supplement of cell-culture medium contains enough of the vitamin to affect the differentiation of cultured keratinocytes derived from epidermis and from other stratified squamous epithelia. The cellular and molecular properties of the cultures are altered when the medium is supplemented with serum from which the vitamin A has been removed by solvent extraction (delipidized serum). Cell motility is reduced, the adhesiveness of cells increases and pattern formation is prevented. In both epidermal and conjunctival keratinocytes, removal of vitamin A leads to the synthesis of a 67 kd keratin characteristic of terminally differentiating epidermis and to much reduced synthesis of the 52 kd and 40 kd keratins typical of conjunctiva. These changes, both cellular and molecular, are reversed by the addition of retinyl acetate to the medium containing delipidized serum. Cell motility and pattern formation are restored, and detachment of the most mature cells from the surface of the stratified epithelium is promoted. Synthesis of the 67 kd keratin is prevented and the synthesis of the 40 and 52 kd keratins is stimulated. The nature of the keratins synthesized is regulated by the concentration of vitamin A, and each cell type adjusts its synthesis differently at a given vitamin concentration.  相似文献   

3.
We have studied the effect of various fractions of foetal bovine serum upon the endogenous degradation of long labelled proteins in cultured MRC5 cells, and upon other cellular functions. Only heat-inactivated serum was capable of suppressing protein degradation to a similar extent to complete serum. Acid-treated and delipidized sera were moderately effective. Albumin on its own was able to replace 40 per cent of the effect of serum, indicating the exogenous protein might compete with endogenous protein for degradation in lysosomes. Albumin was not capable of supporting DNA synthesis. Dialysed serum showed an age-related effect suppressing protein degradation to a lesser extent and being less effective in supporting DNA synthesis or cellular proliferation in aged cells. All the effects noted were related to lysosomal protein degradation. Serum diffusate did not suppress protein degradation.  相似文献   

4.
Summary Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 μM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.  相似文献   

5.
Somatic cell hybrids were obtained with electric pulse by fusion of human epithelial HeLa cells derived from a carcinoma of the uterine cervix and mouse fibroblasts 3T3.4E, deficient in thymidine kinase. Hybrids were selected and propagated in HAT media; some experiments were carried out in medium with delipidized serum. The hybrid cells were characterized by indirect immunofluorescence with a biotin-streptavidin system using a panel of nine monoclonal antibodies specific for membrane and cytoplasmic antigens of parental cells: intermediate filaments (keratins and vimentin), HLA class 1 (beta 2-microglobulin), cell activation (EGF and transferrin receptors) and cellular adhesion (fibronectin and laminin). All of these antigens were expressed in HeLa cells cultured in conventional medium or with delipidized serum. Conversely mouse fibroblasts contained only vimentin, fibronectin and laminin. All the parental antigens were present in first passage hybrid cells cultured in conventional medium. Vimentin, fibronectin and laminin were maintained in fourth passage hybrids whereas keratins, beta 2-microglobulin, EGF and transferrin receptors were no longer detected. When propagated in medium with delipidized serum, hybrid cells re-expressed these antigens after 5 days of culture. These findings suggest that the reexpression of HeLa cell antigens in hybrid cells was related to deficiency in vitamin A.  相似文献   

6.
Culture in the presence of delipidized serum (i.e., in the absence of vitamin A) has been shown to allow terminal differentiation of human keratinocytes, both in terms of morphological appearance and in terms of 67 kD keratin polypeptide synthesis (Fuchs, E & Green, H, Cell 25 (1981) 617) [2]. Culture at the liquid-air interface is known to induce morphological differentiation in a variety of culture systems designed for keratinocytes (Pruniéras, M et al., J invest dermatol 81 (1983) 28s) [3]. We report here that human keratinocytes grown on a dermal equivalent (or lattice) in the presence of total serum are able to express the 67 kD keratin polypeptide, provided that the culture is raised at the liquid-air interface. Loss of contact with air results in switching off this synthesis.  相似文献   

7.
L cells were cultivated in test medium which contained 14C-sodium acetate, and the amount of labeled digitonin-precipitable sterol was assayed in medium and cells. Increasing concentrations of whole serum in the medium had two effects: depressed cellular synthesis and enhanced release of synthesized sterol from the cells. In experiments with delipidized serum containing unesterified cholesterol, cellular sterol synthesis decreased as free cholesterol concentration in the medium increased. In other experiments using medium containing increasing lecithin concentration and no exogenous sterol, the concentration of lecithin markedly influenced the distribution of synthesized sterol between the cells and the medium which then directly influenced the amount of sterol synthesized. These experiments indicate that cell sterol synthesis is regulated by internal levels of free sterol. This, in turn, is a function of cellular sterol flux which is regulated by the concentration and composition of serum lipoprotein in the medium.  相似文献   

8.
Preparation of delipidized serum protein for use in cell culture systems   总被引:22,自引:0,他引:22  
Summary A rapid procedure for the preparation of delipidized serum protein is described. The delipidized protein can be used for the maintenance and growth of tissue culture cells in a lipid-free environment. The extraction procedure greatly reduces all serum lipid classes and the delipidized protein supports the growth of a variety of cells in culture. This research was supported in part by USPHS research grants HL-09103 and HL-16058 from the National Heart and Lung Institute and RR-05540 from the Division of Research Resources. This work was done during tenure as Established Investigator of the American Heart Association (G.H.R.).  相似文献   

9.
The polyene antibiotic filipin was used to visualize the presence and distribution of cholesterol in the plasma membrane of glutaraldehyde-fixed human chronic lymphocytic leukemia (CLL) cells. Both compactin (ML-236B), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and 25-hydroxycholesterol reduced the content of filipin-cholesterol complexes in the plasma membrane of CLL cells grown in media supplemented with either 15% delipidized horse serum or 15% normal (whole) horse serum. The reduction due to compactin was reversed by the concomitant addition of mevalonolactone. The ability of compactin to reduce the relative cholesterol content (as judged by filipin labeling) in CLL cells grown in lipoprotein-containing (normal) serum suggest that either CLL cells are different from other cells in that they predominantly utilize endogenously synthesized cholesterol for incorporation into the plasma membrane, or that a separate pool of endogenously synthesized cholesterol provides cholesterol for the plasma membrane.  相似文献   

10.
Human hepatoma HepG2 cells were used to demonstrate coordinate regulation of three enzymes of cholesterol synthesis under a variety of conditions. Addition of either delipidized serum and mevinolin or low density lipoprotein, 25-hydroxycholesterol, or mevalonic acid to HepG2 cells resulted in rapid changes both in the levels of the mRNAs and in the rates of synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and farnesyl pyrophosphate synthetase (prenyltranferase). In all cases, the changes in mRNA levels were paralleled by changes in the rates of specific protein synthesis. Pulse-chase techniques were used to determine the half-lives of all three proteins. Addition of low density lipoprotein to the media during the chase increased the rate of degradation of HMG-CoA reductase 4.6-fold but had no affect on the half-lives of HMG-CoA synthase or prenyltransferase. Therefore, we conclude that the coordinate regulation of these three enzymes under a variety of conditions occurs at the level of enzyme synthesis and not at the level of protein stability.  相似文献   

11.
Retinol at concentrations of 10(-6) and 10(-5) M stimulated growth of bovine aortic endothelial cells maintained in Eagle's MEM supplemented with delipidized serum. In addition to retinol, retinal, retinoic acid, and retinyl acetate were also growth stimulatory. At very low inoculum densities (4-40 cells/cm2) the growth promoting effect could be demonstrated only in the presence of conditioned medium from macrophage-like culture P388D1. When added to media containing whole (nondelipidized) serum, retinol was growth inhibitory at 10(-6) and 10(-5) M concentrations.  相似文献   

12.
The effect of a Fibroblast Growth Factor (FGF) on the initiation of DNA synthesis in sparse populations of BALB/c 3T3 cells maintained quiescent in the presence of various serum concentrations has been investigated. The initiation of DNA synthesis, as measured by 3H-thymidine incorporation, is greatest in cultures maintained quiescent in the presence of 0.8% serum. Under these conditions, the cells are on the border between quiescence and growth. The minimal effective dose of FGF needed to increase DNA synthesis is 0.01 ng/ml and plateau values are obtained between 2.5 and 5 ng/ml. At plateau concentrations, FGF is 65% as effective as saturating concentrations of serum in the stimulation of DNA synthesis. When dexamethasone and insulin are present, FGF was 82% as effective. In contrast, cultures maintained in the presence of lower serum concentrations (0.2% and 0.4%) are much less responsive to the FGF. This can be attributed to the lack of supplemental factors which make the cells maximally responsive to growth stimulation and to degenerative changes that take place in the cells. Insulin and the glucocorticoid, dexamethasone, potentiated the response to FGF and delayed the degeneration of cells maintained in low serum.  相似文献   

13.
We compared the ability of dialysed fetal bovine serum and of combinations of purified growth-promoting factors such as insulin, epidermal growth factor (EGF), vasopressin, fibroblast-derived growth factor and antitubulin agents to stimulate DNA synthesis in 3T3 cells maintained in the absence or presence of inorganic phosphate (Pi). When DNA synthesis was stimulated by serum in the absence of Pi the level induced was 70% of that observed in Pi-containing medium. In contrast, combinations of growth-promoting factors in the absence of Pi stimulated less than 8% of the DNA synthesis which they induced in complete medium. Addition of as little as 50 μM Pi fully restored the ability of the factors to stimulate DNA synthesis. Cells stimulated by purified mitogens in the absence of Pi became blocked in early G1, and for up to 48 h the block was reversible by readdition of pi. The effectiveness of dialysed serum to stimulate DNA in the absence of Pi suggest that dialysed serum might contain a component capable of supplying Pi to support DNA synthesis. Indeed, delipidization of serum by solvent extraction resulted in loss of ability to stimulate DNA synthesis in the absence of pi, but delipidized serum stimulated DNA synthesis virtually, as well as dialysed serum in the presence of Pi. Previous conclusions suggesting that Pi is not essential for DNA synthesis appear to require re-evaluation.  相似文献   

14.
Addition of serum to resting cultures of Swiss mouse 3T3 cells causes an immediate multiple phosphorylation of 40S ribosomal protein S6. After 60 min of stimulation, changing to medium containing no serum led to the net dephosphorylation of S6. During this same period, a second protein, as yet unidentified, became increasingly phosphorylated. Incubation of cells with cycloheximide prior to the addition of serum almost completely blocked the activation of protein synthesis. There was no effect on the serum-induced phosphorylation of S6. If cells were stimulated in the presence of cAMP phosphodiesterase inhibitors theophylline or SQ 20006, both S6 phosphorylation and the activation of protein synthesis were inhibited. Stimulation of cells with serum also led to an immediate drop in total intracellular cAMP levels. This was blocked by prostaglandin E1 (PGE1), which caused a 10 fold increase in total intracellular cyclic AMP. However, PGE1 had no effect on protein synthesis or S6 phosphorylation.  相似文献   

15.
As a model cell culture system for studying polyoma-mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble portein, corresponding to an increase of approximately 100-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA. In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a 2-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin E1, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.  相似文献   

16.
Tissue culture cells require lipid which must be provided exogenously or synthesized via endogenous pathways. The exogenous supplies can be largely removed by growing cells in medium containing delipidized serum. Pathways for synthesis of lipid can then be blocked at three steps: (1) fatty acids by removal of biotin, an essential coenzyme; (2) phosphatidylcholine and sphingomyelin by deleting choline from the growth medium; and (3) cholesterol by inhibiting HMG-CoA reductase with 25-hydroxycholesterol. Sustained proliferation is prevented when lipid synthesis is blocked at any one of these steps. Cell proliferation resumes upon restoring synthesis with biotin, choline, or mevalonate (the product of the HMG-CoA reductase reaction) or by providing the lipid end products oleic acid or cholesterol. Using a combined cytophotometric-autoradiographic analysis to determine cell cycle distributions we have demonstrated that prereplicative (G1) cell cycle arrests develop in parallel with the proliferative inhibition. Each of the G1 arrests can be reversed by restoring the synthetic pathways or their lipid products. These observations suggest a causal relationship between the supply of lipids and passage through G1.  相似文献   

17.
18.
Biotin or a serum lipid extract stimulated proliferation of G1 arrested Rous sarcoma virus-transformed BHK cells in modified Eagle's MEM (BM). The cells could be maintained continuously in BM plus biotin (BMB), but not in BM plus serum lipid extract (BM X L). Avidin inhibited growth stimulation when added to BMB, but did not inhibit growth when added to BM X L. 14C-acetate incorporation into total cellular lipids was stimulated in BMB, but not in BM. Thin-layer chromatography of the labeled cellular lipid extract indicated that relatively large amounts of 14C-acetate were incorporated into phosphatidylserine and little into the other major phospholipids. In the neutral lipids, the largest amount of incorporation was in cholesterol. G1 arrested cells multiplied rapidly in BM supplemented with dialyzed serum (BM X DS), but they did not multiply in BM with delipidized serum (BM X DLS). The addition of biotin or serum lipid extract to BM X DLS stimulated growth. Growth stimulation in BM X DLS by biotin was inhibited by avidin, but avidin had no effect on growth stimulation by serum lipid extract. Biotin stimulated additional multiplication in BM X DS and avidin inhibited this additional growth stimulation. These results suggest that growth stimulation requires lipids supplied by serum lipids or by de novo synthesis stimulated by biotin. In the absence of serum, the stimulation of the synthesis of growth factor(s) by biotin are also required for continuous multiplication.  相似文献   

19.
When isolated confluent corneal endothelial cells were cultured in delipidized serum, a marked reduction in collagen production was observed. Supplementation of such cultures with vitamin A as either retinol or retinoic acid at concentrations of 10?6–10?7, M was capable of significantly increasing collagen production. In addition, when cultured in normal (non-delipidized) serum, both retinol and retinoic acid were capable of further increasing collagen production by corneal endothelial cells. Such augmentation of collagen production was relatively specific as total protein synthesis was not altered to the same extent, nor was it merely a reflection of changes in total cell number, as such cell numbers were similar in all treatment groups.  相似文献   

20.
This work was undertaken to assess the kinetics of boronated porphyrin cellular uptake, which has been reported to occur by way of the low-density lipoprotein receptors. Because of current interest in the use of boronated porphyrins in boron neutron capture therapy of tumors, this pathway was investigated for the cellular uptake of a boronated porphyrin (tetrakis-carborane-carboxylate, esters of 2,4-bis (alpha,beta-dihydroxyethyl) deuteroporphyrin IX). Boron uptake occurred even without low-density lipoprotein in the culture medium. Pre-incubation of V-79 Chinese hamster cells for 24 h in medium containing delipidized fetal bovine serum markedly increased the subsequent uptake of boron when compared with cells pre-incubated with medium containing 10% fetal bovine serum. The increased uptake was characterized by greater affinity for boronated porphyrin, compared to cells pre-incubated in 10% fetal bovine serum. Twenty-four hour preincubation of cells with increasing concentrations of LDL added to delipidized medium suppressed the up-regulation of the boron level. In contrast, incubation with added acetylated LDL did not prevent the up-regulation of boron uptake. Positive cooperativity was demonstrated by Hill and Scatchard plots. It is concluded that uptake of boronated porphyrin is characterized by positive cooperativity, that its uptake is markedly enhanced when preincubated in delipidized serum, and that significant uptake occurs even in the absence of low density lipoprotein in the medium. These data suggest a novel way for enhancing uptake of boron (and perhaps other agents) into tissues using carrier porphyrins, by increasing the number and/or affinity of cellular LDL receptors.  相似文献   

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