Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture.

The fibrinolytic activity of cells in culture varied with the type of serum employed in the growth medium. Degradation of iodinated fibrin occurred slowly when Rous sarcoma virus-transformed chick embryo fibroblasts were grown in medium containing fetal bovine serum (FBS), and rapidly when chicken serum was employed. This difference reflected the low plasminogen and high inhibitor content of FBS. The inhibitors were found to be serum macromolecules that were precipitated with ammonium sulfate or polyethylene glycol, and were inactivated by boiling or upon exposure to acidic conditions. No inhibitor activity was detected in fetuin, one of the major proteins present in FBS. Acidified FBS was similar to chicken serum in that both supported high rates of cell-mediated fibrinolytic activity. Although virally transformed hamster, mouse and chicken cells grew well in acid-treated FBS, their normal counterparts did not. Apparently, acifification resulted in the formation of materials that were toxic to normal cells. These agents rapidly blocked cellular DNA synthesis.     

Serum and plasma stimulate prostaglandin production by alveolar macrophages

Fetal bovine serum (FBS) stimulated rabbit alveolar macrophages to synthesize prostaglandins (PG) and release lysosomal enzymes. This stimulatory action was not entirely due to the effect of foreign protein in FBS, since rabbit serum and plasma, both homologous and autologous, also induced release of PGs and lysosomal enzymes. Rabbit serum and plasma are less effective than FBS as a stimulus for PG release, with rabbit serum being more potent than plasma at the same concentration. Bovine serum albumin elicited a dose-dependent increase of arachidonic acid release by macrophages, but not of PG production. Hence, the fatty acid "trapping" effect of albumin in serum and plasma is not responsible for the PG stimulation. The PG stimulating factors were stable at 56 degrees C for 30 min., but lost half the activity after heating at 100 degrees C for 10 min. Gel permeation chromatography of FBS showed several peaks of PG stimulating and arachidonic acid releasing activity. The molecular weight of the major one (150,000 daltons) is similar to that of immunoglobulin G. Rabbit IgG, when added to the macrophage culture, stimulated release of arachidonic acid and PGs. However, the major stimulatory effect in serum or plasma is not all due to IgG, since removal of IgG by a Protein A-agarose column did not remove the stimulatory effect of FBS and rabbit serum. The possibility of other factors, such as complement fragments, is discussed.     

Macrophage-mediated fungistasis: requirement for a macromolecular component in serum

Peritoneal macrophages from Mycobacterium bovis- or Toxoplasma gondii-infected mice cultured in vitro in Dulbecco's medium containing 10% fetal bovine serum (FBS) and endotoxin stopped replication of Cryptococcus neoformans for 30 hr, whereas yeast cells cultured alone reproduced with a 3.0-hr doubling time. Without at least 5% FBS, macrophage fungistasis was poor. FBS without macrophages enhanced the growth rate of cryptococci. Macrophages preincubated in vitro for 24 hr without serum became fungistatic when challenged with cryptococci in medium with FBS but were not fungistatic without FBS. Macrophages preincubated in medium with FBS were never subsequently fungistatic. Dialyzed, heated (56 degrees C, 30 min), or delipidated FBS supported macrophage fungistasis, whereas FBS heated at 70 degrees C for 30 min did not. FBS contained no measurable opsonic activity for C. neoformans. Inclusion of endotoxin and/or murine IFN-gamma over wide concentration ranges did not substitute for FBS. Ultrafiltration estimation of FBS activity localized to 50 to 150 Kd. By gel filtration chromatography, FBS activity ran in the 25 to 100 Kd range. Dye-ligand affinity chromatography on Cibacron blue agarose gel dissociated the FBS activity from the albumin and lipoprotein fractions. Anion-exchange chromatography on DEAE-Sephacel revealed activity in the first fraction eluting at low ionic strength, pointing to a protein(s) with an isoelectric point toward neutral. Activated macrophages can prevent microbial replication within host tissues; the local environment is critical for fulfillment of this important physiologic function. These results point to a macromolecular factor(s) present in serum that is essential for full fungistatic capability of activated macrophages.     

Effect of serum on organogenesis of the rat testis in vitro

Summary It was observed previously that primordia of fetal rat testes when explanted in vitro in a synthetic medium at the outset of sexual differentiation differentiate seminiferous cords during the following days, but that the addition of 15% fetal bovine serum prevents this morphogenesis. In the present study, human, horse, bovine calf, and rat sera were shown to exert the same effect. Very low concentrations of human or fetal bovine serum (0.5 or 1%) were sufficient to produce the serum effect, which was only slightly reduced when the serum was heated. The serum activity was not removed by dialysis (membrane cut-off 15 000), but it disappeared after treatment with trichloroacetic or perchloric acids or after trypsin digestion. Partial purification of the active factor(s) from human serum was achieved by successive gel filtration, affinity chromatography, and ion exchange chromatography. Analysis of the active fractions by electrofocusing and immunoelectrophoresis placed the activity within the α globulin group. Among a series of purified serum proteins tested, α₂-HS-glycoprotein was found to exhibit the serum effect, though this activity was heat labile.     

Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.     

Antisera reactive directly to estrone sulfate.

Derivatives of estrone were prepared and linked to bovine serum albumin or its methyl-esterified form to produce immunogens which were effective in raising antisera to estrone sulfate. The most effective was estrone-3-methylphosphonothioate, electrostatically complexed with methylated bovine serum albumin. The ionically combined hapten functioned as an antigenic determinant as do covalently bound haptens when administered to sheep in emulsions with Freund's complete adjuvant. Estrone-3-phosphate covalently or electrostatically linked to bovine serum albumin also produced antisera reactive to estrone sulfate. Estrone sulfate itself, after electrostatically complexing to methylated bovine serum albumin and administration with Freund's complete adjuvant to sheep, was ineffective in producing antisera. The sera which had workable titres to estrone sulfate showed considerable cross-reaction with free estrone but was otherwise highly specific with little or no reaction with other steroid sulfates, glucosiduronates or other free steroids. Radioimmunoassay curves using [6,7-3H]-estrone sulfate were highly sensitive and were effective in the range of 5-250 pg estrone sulfate.     

Serum and plasma stimulate prostaglandin production by alveolar macrophages

Fetal bovine serum (FBS) stimulated rabbit alveolar macrophages to synthesize prostaglandins (PG) and release lysosomal enzymes. This stimulatory actions was not entirely due to the effect of foreign protein in FBS, since rabbit serum and plasma, both homologous and autologous, also induced release of PGs and lysosomal enzymes. Rabbit serum and plasma are less effective than FBS as a stimulus for PG release, with rabbit serum being more potent than plasma at the same concentration. Bovine serum albumin elicited a dose-dependent increase of arachidonic acid release by macrophages, but not of PG production. Hence, the fatty acid “trapping” effect of albumin in serum and plasma is not responsible for the PG stimulation. The PG stimulating factors were stable at 56°C for 30 min., but lost half the activity after heating at 100°C for 10 min. Gel permeation chromatography of FBS showed several peaks of PG stimulating and arachidonic acid releasing activity. The molecular weight of the major one (150,000 daltons) is similar to that of immunoglobulin G. Rabbit IgG, when added to the macrophage culture, stimulated release of arachidonic acid and PGs. However, the major stimulatory effect in serum or plasma is not all due to IgG, since removal of IgG by a Protein A-agarose column did not remove the stimulatory effect of FBS and rabbit serum. The possibility of other factors, such as complement fragments, is discussed.     

Differential cytopathogenicity accompanyingMycoplasma pneumoniae infection of human lung fibroblasts maintained in newborn bovine serum or fetal bovine serum

Summary MRC-5 human lung fibroblasts maintained in Eagle's basal medium (BME) with either 10% fetal bovine serum (FBS) or 10% newborn bovine serum (NBS) did not respond identically to infection byMycoplasma pneumoniae. Fibroblasts grown in NBS did not develop any cytopathic effect (CPE) when infected withM. pneumoniae, whereas those maintained in FBS developed a pronounced CPE. There was also a difference in sensitivity to infection for fibroblasts maintained in the two sera before the infection. Fibroblasts maintained in NBS, then transferred to FBS 48 h before infection, were still less sensitive toM. pneumoniae infection than cells maintained constantly in FBS.Mycoplasma pneumoniae attached comparably to the fibroblasts grown in the two sera, so the differences in CPE development could not be attributed to differences in mycoplasma attachment. Measurements of DNA, RNA, and protein syntheses of the fibroblasts grown in NBS and FBS indicate that the cells in NBS were growing more rapidly than those in FBS. A determination of the doubling times shows that the doubling time of cells in NBS was 44 h, whereas that of cells in FBS was 51 h. Polyacrylamide gel electrophoresis of samples of NBS and FBS showed significant differences in serum protein composition. The NBS had several protein bands that were lacking in the FBS. This study demonstrates the importance of serum effects in the study ofM. pneumoniae infection. This work was supported in part by Public Health Service Grant AI 17795 from the National Institute of Allergy and Infectious Diseases, Bethesda, MD.     

Effects of serum type on growth and permeability properties of cultured endothelial cells

Serum is frequently added to defined basal media as a source of certain nutrients and macromolecular growth factors essential for cell growth. The many different sera commercially available may not be equally suitable for all cell types. The effects of four sera, fetal bovine serum (FBS), calf bovine serum (CS), equine serum (ES-1), and plasma-derived equine serum (ES-2), on growth and permeability properties of cultured porcine endothelial cells were determined. The rate of DNA synthesis, measured as [3H]thymidine incorporation, reached a peak at around 24 h, regardless of serum type, and was most marked with ES-1- or ES-2-treated cells. However, when estimated by total DNA, FBS, CS, or ES-1 treatment resulted in greater cell proliferation than ES-2. Based on protein synthetic rate and total cell protein, both FBS and CS appeared to be most growth supporting. At 72 h after cell plating, albumin passage across cultured endothelial monolayers was elevated in ES-1- and ES-2-treated cells compared with FBS- or CS-treated cells. "Leaky" cell monolayers were most marked with ES-1-treated cells. Cells grown in ES-2- and particularly in ES-1-enriched media were larger and more spindle-shaped compared with the typical cobblestone appearance of cells cultured in media enriched with either FBS or CS. These data suggest that CS, but not ES-1 or ES-2, is an excellent substitute for FBS to support desirable growth properties of macrovascular endothelial cells in culture.     

Optimization of cryoprotectants for cryopreservation of rat hepatocyte

Rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing either fetal bovine serum (FBS), glycerol, dimethyl sulfoxide (DMSO), sucrose or a mixture of these as a cryoprotectant. The best survival was with 10% (v/v) DMSO containing 30% (v/v) FBS using 5 x 10(5) hepatocytes ml(-1) at -70 degrees C for 5 d on type I collagen-coated dishes. After thawing, the cell viability was 81% determined by the MTT-test. The cryopreserved hepatocytes had the capacity of albumin synthesis similar to hepatocytes without cryopreservation. This result shows that cryopreservation of rat hepatocyte can be used for the evaluation of hepatic functions.     

Expression of cell adhesion molecule and albumin genes in primary culture of rat hepatocytes

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin alpha-1 mRNA. The changes in mRNA levels of liver cadherin and integrin alpha-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell-cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Change of Morphology and Cytoskeletal Protein Gene Expression during Dibutyryl cAMP-induced Differentiation in C6 Glioma Cells

Elevation of the intracellular cAMP level induces morphological changes of astrocyte-like differentiation in C6 glioma cells. Such changes may be accompanied with expression of cytoskeletal protein genes. We therefore analyzed morphological changes after a treatment with dibutyryl cAMP (dbcAMP) and then assessed the expression of cytoskeletal protein genes by a quantitative real-time polymerase chain reaction. The cell number remained unaltered upon incubation with 1 mM dbcAMP in medium supplemented with 0.1% fetal bovine serum (FBS), whereas the number and lengths of processes increased, when compared with those of cells incubated in medium supplemented with 0.1% or 10% FBS only. The amounts of β-actin, γ-actin, and β-tubulin mRNAs in C6 cells, but not α-tubulin mRNA, increased during the early proliferation in DMEM containing 10% FBS. The expression of cytoskeletal protein genes decreased when incubated with 0.1% FBS or 1 mM dbcAMP in 0.1% FBS, compared with those of cells cultured in 10% FBS. These results indicated that, during the early proliferation in normal culture condition, the expression of cytoskeletal protein genes in C6 cells, except α-tubulin, increased, while in differentiating or differentiated C6 glioma cells, cAMP-induced morphological changes were not accompanied with elevation of gene expression for cytoskeletal proteins, such as actin and tubulin.     

Estrogen-dependent modification of ribosomal proteins. Effects of estrogen withdrawal on the distribution of constitutive and hormonally regulated mRNAs

Estrogen-dependent modification of ribosomal proteins during induction of egg-yolk protein synthesis in avian liver was examined in vivo and in cultured hepatocytes. Modification of two proteins of the 40S ribosomal subunit was detected in vivo, within 40 min of injection of hormone. One of the proteins was identified as S6 and the other, tentatively, as S3a. Estrogen treatment resulted in the appearance of multiple, phosphorylated forms of S6 and a shift in electrophoretic mobility of the other protein that was consistent with its dephosphorylation. The steady state achieved within 2 h of injection could be maintained for up to 2 weeks when the hormone was administered from silastic implants. Removal of the implants resulted in a return to the preinduction state within 20-40 min. Similar modifications were induced in hepatocytes maintained in defined medium, with 17 beta-estradiol as the only hormonal supplement. In order to check on the possibility that the modifications observed could selectively influence mRNA utilization, the cytoplasmic distributions of two abundant mRNAs were monitored during the first few hours following withdrawal. One of these was serum albumin mRNA, the levels of which are unaffected by estrogen. The other was very-low-density apolipoprotein II mRNA which specifies a major egg-yolk protein. The synthesis of this mRNA is absolutely dependent on estrogen and its half-life is also markedly affected by the hormone.     

Activation of delta5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins.

The Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ⁵-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.     

Hormonal Regulation of Leptin mRNA Expression and Preadipocyte Recruitment and Differentiation in Porcine Primary Cultures of S-V Cells

The hormonal regulation of leptin mRNA expression and the association between leptin expression and adipocyte differentiation were examined in primary cultures of porcine S-V cells with Northern blot and immunocytochemical analysis. Seeding for 3 days with fetal bovine serum (FBS) with varying levels of dexamethasone (Dex) increased levels of leptin mRNA in a dosedependent manner in parallel with increases in the proportion of preadipocytes (AD-3 positive cells; AD-3, a preadipocyte marker). Six-day treatment with 10 or 850 nM insulin after FBS+Dex treatment resulted in a similar increase in leptin mRNA expression and morphological differentiation. However, significantly lower levels of leptin mRNA and smaller fat cells were observed in cultures treated with 1 nM insulin or 10 nM insulin-like growth factor-I (IGF-I). Dex-induced increases in leptin mRNA levels and AD-3 cell numbers were blocked completely by the addition of transforming growth factor-β (TGF-β) to FBS+Dex-treated cultures. However TGF-β significantly increased fat cell size and leptin mRNA expression when added to ITS (insulin, 850 nM; transferrin, 5 μg/ml; and selenium, 5 ug/mL) treated cultures during the lipid-filling stage. When added with FBS+DEX for the first 3 days, growth hormone (GH) did not influence the Dex-induced increase in AD-3 cells and leptin mRNA expression, but GH reduced leptin mRNA levels when added with insulin for 6 days after FBS+Dex. These results demonstrated that regulation of leptin mRNA expression by Dex, insulin, IGF-I, TGF-β, and GH may be associated with changes in preadipocyte number and fat cell size.