scholarly journals Changes in sterol biosynthesis accompanying cessation of glial cell growth in serum-free medium

1980 ◽  
Vol 192 (2) ◽  
pp. 709-717 ◽  
Author(s):  
William A. Maltese ◽  
Beverly A. Reitz ◽  
Joseph J. Volpe
Keyword(s):  
Fatty Acid ◽  
Reductase Activity ◽  
Calf Serum ◽  
Sterol Synthesis ◽  
Foetal Calf Serum ◽  
Quiescent State ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Coa Reductase

C-6 glioma cells, grown in medium supplemented with 5% delipidated foetal calf serum, were induced to enter a quiescent state by removing serum from the medium. Within 24h there was a 75–80% decline in the rate of incorporation of [14C]acetate or 3H2O into digitonin-precipitable sterols. Experiments with [3H]mevalonolactone as a labelled sterol precursor suggested that the decline in sterol synthesis was regulated primarily at a point in the pathway before the formation of mevalonate. The specific activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase decreased sharply in conjunction with the decline in sterol synthesis in the serum-free cultures; however, the activity of acetoacetyl-CoA thiolase was altered only slightly. The magnitude of the initial decline in reductase activity was not affected when 50-mm-NaF was included in the preincubation and assay buffers to prevent activation of physiologically inactive enzyme. However, after 6h of serum deprivation the decline in 3-hydroxy-3-methylglutaryl-CoA reductase activity was due to a decrease in the amount of latent activity. The sterol concentration in C-6 cells was unchanged after 24h in serum-free medium, although a 20% decrease in the sterol/fatty acid molar ratio occurred as a result of a small increase in the fatty-acid concentration. Incorporation of 3H2O into fatty acids was inhibited in the serum-deprived glial cells; however, this inhibition developed more slowly and was not as pronounced as the diminution in sterol synthesis. The results suggest that in C-6 glia, which resemble the glial stem cells of the developing brain, the decreased demand for membrane sterols in the quiescent state results in a decline in sterol synthesis, mediated primarily through co-ordinate changes in the activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.

Exogeneous factors are not necessary in attachment, spreading and polarization of hela cells

1978 ◽  
Vol 36 (2) ◽  
pp. 310-311
Author(s):  
W. Liebrich
Keyword(s):  
Hela Cells ◽  
Calf Serum ◽  
Glass Tube ◽  
Serum Free Medium ◽  
Nacl Solution ◽  
Free Medium ◽  
Minimum Essential Medium ◽  
Serum Free ◽  
All Solutions ◽  
Glass Support

HeLa cells were grown for 2-3 days in EAGLE'S minimum essential medium with 10% calf serum (S-MEM; Seromed, München) and then incubated for 24 hours in serum free medium (MEM). After detaching the cells with a solution of 0. 14 % EDTA and 0. 07 % trypsin (Difco, 1 : 250) they were suspended in various solutions (S-MEM = control, MEM, buffered salt solutions with or without Me++ions, 0. 9 % NaCl solution) and allowed to settle on glass tube slips (Leighton-tubes). After 5, 10, 15, 20, 25, 30, 1 45, 60 minutes 2, 3, 4, 5 hours cells were prepared for scanning electron microscopy as described by Paweletz and Schroeter. The preparations were examined in a Jeol SEM (JSM-U3) at 25 KV without tilting.The suspended spherical HeLa cells are able to adhere to the glass support in all solutions. The rate of attachment, however, is faster in solutions without serum than in the control. The latter is in agreement with the findings of other authors.

Cell surface heparan sulphate and adhesive property of sublines of rat ascites hepatoma AH7974

1988 ◽  
Vol 90 (4) ◽  
pp. 683-689 ◽  
Author(s):  
A. Kimura ◽  
T. Kawaguchi ◽  
T. Ono ◽  
A. Sakuma ◽  
Y. Yokoya ◽  
...  
Keyword(s):  
Cell Surface ◽  
Culture Medium ◽  
Heparan Sulphate ◽  
Soluble Form ◽  
Foetal Calf Serum ◽  
Serum Free Medium ◽  
Free Medium ◽  
Ascites Hepatoma ◽  
Serum Free ◽  
Rat Ascites Hepatoma

Two variants (74AD and 74FL) established from rat ascites hepatoma AH7974 were examined for the production of glycosaminoglycans in culture. There was no difference between the adhesive (74AD) and the floating (74FL) variants in quantity of glycosaminoglycans produced by their cultivation in minimum essential medium supplemented with 10% foetal calf serum. However, they were distinctly different in the distribution patterns of heparan sulphate. In 74FL, about 70% of total heparan sulphate was found in the culture medium in soluble form, whereas in 74AD, only 7% was found in the medium and the rest was in the cell-substratum complex. In a serum-free medium, 74AD cells grew without adhering to the substratum. After cultivation, more than 90% of total heparan sulphate was found in the cell-associated fractions and the rest in the substratum fractions. No heparan sulphate was detected in the culture medium. On the other hand, 74FL cells released heparan sulphate to the serum-free medium as much as to the serum-containing medium. The increase in amount of heparan sulphate in the culture medium of 74FL cells was supposed to be caused by failure of the cells to deposit heparan sulphate at the cell surface and not caused by increased production. Cell-substratum adhesion mechanisms involving cell surface heparan sulphate (heparan sulphate proteoglycan) and some serum intermediate(s) are discussed for 74AD cells.

Stimulierung von Kulturen embryonaler Rattenzellen durch Kälberserum, III / Stimulation of Embryonic Rat Cells in Culture by Calf Serum, III

1972 ◽  
Vol 27 (11) ◽  
pp. 1399-1404 ◽  
Author(s):  
Michael Humpel ◽  
Werner Frank
Keyword(s):  
Rna Synthesis ◽  
De Novo ◽  
Calf Serum ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Uridine Uptake ◽  
Insoluble Material ◽  
Turn Over ◽  
Precursor Molecules

Embryonic rat cells which have been stopped in G1-phase of their cell cycle by incubation in serum-free medium can be triggered by the addition of calf serum.Expamination of uridine uptake and RNA synthesis after stimulation gave the following results:1) Uridine uptake into the acid soluble pool is increased within a few minutes.2) 2 hours after the addition of serum cells incorporate twice as much 3H-uridine into the acid insoluble material as do cells in serum-free medium; this is not the result of a higher rate of RNA synthesis but is due to an increased uridine uptake.As demonstrated by isolation of RNA and electrophoresis in polyacrylamide gels a significant de novo synthesis can be detected 4 hours after stimulation. Sedimentation coefficients of these RNA species are between 28S and 18S, and 18S and 4/5S, resp.; they turn over quite rapidly as was demonstrated by chase experiments.3) When cells are grown for 2 hours in medium containing 3H-uridine, little label can be detected in the ribosomal 18S- and 28S-RNA. Radioaktivity in these species is, however, strongly increased after another incubation period of 2 hours in culture medium without 3H-uridine. This indicates that precursor molecules ars synthetized and subsequently degrated into 28S- and 18S-rRNA.

scholarly journals Stimulierung von Kulturen embryonaler Rattenzellen durch eine Proteinfraktion aus fötalem Kälberserum / Stimulation of Embryonic Rat Cells in Culture by a Protein Fraction Isolated from Fetal Calf Serum

1971 ◽  
Vol 26 (10) ◽  
pp. 1045-1048 ◽  
Author(s):  
Dieter F. Hülser ◽  
Werner Frank
Keyword(s):  
Cell Cycle ◽  
Culture Medium ◽  
Fetal Calf Serum ◽  
Surface Membrane ◽  
Calf Serum ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Membrane Potential Difference ◽  
Stimulation Of

Normal embryonic rat cells incubated in serum-free medium accumulate in G1-phase of the cell cycle. On addition of a growth-stimulating protein isolated from fetal calf serum they are triggered to proceed through the cycle, and they resume DNA-synthesis 15 to 20 hours later. In this paper it is demonstrated that the surface membrane potential difference (PD) decreases immediately after changing serum-free medium against culture medium containing either calf serum or the isolated serum protein; the original PD is restored 2 to 3 hours later. Serumprotein without growthstimulating activity does not affect the PD.A permanent rat cell line which grows independently of serum also has been tested. The PD of these cells is not significantly influenced by calf serum.

Generation of Dendritic Cells from Human Chronic Myelomonocytic Leukemia Cells in Fetal Calf Serum-Free Medium

2000 ◽  
Vol 38 (5-6) ◽  
pp. 577-586 ◽  
Author(s):  
Leopold Oehler ◽  
Andrea Berer ◽  
Felix Keil ◽  
Georg Weinländer ◽  
Margit König ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Chronic Myelomonocytic Leukemia ◽  
Leukemia Cells ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Myelomonocytic Leukemia

Stimulierung von Kulturen embryonaler Rattenzellen durch Kälberserum / Stimulation of Embryonic Rat Cells in Culture by Calf Serum

1972 ◽  
Vol 27 (5) ◽  
pp. 562-566 ◽  
Author(s):  
Johanna Grimm ◽  
Werner Frank
Keyword(s):  
Synthetic Medium ◽  
Calf Serum ◽  
Lag Phase ◽  
Double Labelling ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Protein Binding Assay ◽  
Adenosine Phosphates ◽  
In Cells

Embryonic rat cells cultured in a synthetic medium are stopped in G1 phase if serum is omitted from the culture medium. On addition of calf serum these cells resume DNA synthesis after a lag phase of 15—20 hours. In the present paper the events concerning the metabolism of adenosine phosphates after stimulation by serum have been examined. The results are the following:1. The concentration of cAMP in cells which have been incubated for 24 hours in serum-free medium is 32 times higher than that in cells growing in medium containing 10 per cent calf serum; the accumulated cAMP is metabolized within 10 min after the addition of 10 per cent calf serum. This has been confirmed by a double labelling technique after preincubation with radioactive adenosine as well as by estimation of cAMP using a protein-binding assay.2. A cAMP-specific phosphodiesterase is activated shortly after stimulation; the enzyme activity in dialysed cell homogenates is increased within 30 min by a factor of 2.3. Cells incubated in serum-free and those in serum-containing medium have equal amounts of ATP. On addition of serum the ATP concentration in cells preincubated in serum-free medium decreases by 27%; the original level is restored 20—30 min later.

scholarly journals Fatty Acid Modification of the Anticancer Peptide LVTX-9 to Enhance Its Cytotoxicity against Malignant Melanoma Cells

Toxins ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 867
Author(s):  
Fengjiao Li ◽  
Saizhi Wu ◽  
Ninglin Chen ◽  
Jingyu Zhu ◽  
Xinxin Zhao ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cytotoxic Activity ◽  
Anticancer Drugs ◽  
Melanoma Cells ◽  
Serum Free Medium ◽  
Free Medium ◽  
Acid Modification ◽  
Anticancer Peptide ◽  
Serum Free ◽  
Fatty Acid Modification

Spider venom is a valuable resource for the development of novel anticancer drugs. In this study, we focused on novel linear amphipathic α-helical anticancer peptide LVTX-9, which was derived from the cDNA library of the venom gland of the spider Lycosa vittata. The cytotoxicity of LVTX-9 against murine melanoma cells in the range of 1.56–200 μM was tested and found to be significantly lower than those of most anticancer peptides reported. Its IC50 was determined to be 59.2 ± 19.8 μM in a serum or 76.3 ± 12.7 μM in serum-free medium. Fatty acid modification is a promising strategy for improving peptide performance. Therefore, to enhance the cytotoxic activity of LVTX-9, fatty acid modification of this peptide was performed, and five different carbon chain length lipopeptides named LVTX-9-C12-C20 were produced. Among them, the lipopeptide LVTX-9-C18 showed the highest cytotoxic activity in relation to B16-F10 cells, whether in a serum or serum-free medium. Most importantly, the cytotoxic activity of LVTX-9-C18 was improved by about 12.9 times in a serum medium or 19.3 times in a serum-free medium compared to that of LVTX-9. Subsequently, assays including scanning electron microscopy, trypan blue staining, lactate dehydrogenase leakage assay, and hemolytic activity could indicate that the potential direct cell membrane disruption is the main mechanism of LVTX-9-C18 to induce cancer cell death. Furthermore, the LVTX-9-C18 also showed strong cytotoxicity in relation to 3D B16-F10 spheroids, which indicates it might be a promising lead for developing anticancer drugs.

VI. Calcium- und Kaliumionen als Cofaktoren

1973 ◽  
Vol 28 (5-6) ◽  
pp. 322-328 ◽  
Author(s):  
Werner Frank
Keyword(s):  
Culture Medium ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
The Other ◽  
Potassium Ions ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Factors ◽  
Serum Free ◽  
Intracellular Concentrations

Embryonic rat cells are stopped in G1-phase when they are incubated in serum-free medium. They can be triggered to proceed through their cell cycle by the addition of two proteins which have been isolated from fetal calf serum. The influence of calcium and potassium ions on the stimulation process has been exam ined: 1. Ca2+ and K+ ions simultaneously must be present in the culture medium in concentrations of at least 5x10-4 ᴍ and 1x10-3 ᴍ, resp. 2. The intracellular concentrations of both ions are increased by the serum factors. 3. The intracellular concentrations of K+ strongly are dependent on the extracellular Ca2+-concentrations; they increase threefold between 5x10-5 and 1x10-3 ᴍ of Ca2+. On the other hand, the intracellular calcium concentrations are not significantly influenced by the potassium content of the culture medium.

Hemoglobin Enhances the Binding of Bacterial Endotoxin to Human Endothelial Cells

1996 ◽  
Vol 76 (02) ◽  
pp. 258-262 ◽  
Author(s):  
Robert I Roth
Keyword(s):  
Endothelial Cells ◽  
Binding Protein ◽  
Bacterial Endotoxin ◽  
Dependent Manner ◽  
Serum Free Medium ◽  
Free Medium ◽  
Human Endothelial Cells ◽  
Serum Factors ◽  
Serum Free ◽  
Lps Binding

SummaryHuman endothelial cells, when incubated with bacterial endotoxin (lipopolysaccharide, LPS), modify their surface in association with prominent production of procoagulant tissue factor (TF) activity. This deleterious biological effect of LPS has been shown previously to be enhanced approximately 10-fold by the presence of hemoglobin (Hb), a recently recognized LPS binding protein that causes disaggregation of LPS and increases the biological activity of LPS in a number of in vitro assays. The present study was performed to test the hypothesis that Hb enhances the LPS-induced procoagulant activity of human umbilical vein endothelial cells (HUVEC) by increasing LPS binding to the cells. The binding of 3H-LPS to HUVEC was determined in the absence or presence of Hb or two other known LPS-binding proteins, human serum albumin (HSA) and IgG. LPS binding was substantially increased in the presence of Hb, in a Hb concentration-dependent manner, but was not increased by HSA or IgG. Hb enhancement of LPS binding was observed in serum-free medium, indicating that there was no additional requirement for any of the serum factors known to participate in the interaction of LPS with cells (e.g., lipopolysaccharide (LPS)-binding protein (LBP) and soluble CD14 (sCD14)). Hb enhancement of LPS binding also was observed in the more physiologic condition of 100% plasma. LPS-induced TF activity was stimulated by Hb, but not by HSA or IgG. In serum-free medium, TF activity was not stimulated under any of the conditions tested. Ultrafiltration of LPS was dramatically increased after incubation with Hb but not with HSA or IgG, suggesting that LPS disaggregation by Hb was responsible for the enhanced binding of LPS to HUVEC and the subsequent stimulation of TF activity.

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