Effect of steroid hormones on vaginal epithelial cells: Anin vitro model for steroid hormone action

Conditions have been standardized to maintain rat vaginal epithelial cellsin vitro with more than 95% viability. Cultured epithelial cells were used to study the effects of normal fetal calf scrum, estradiol and progesterone on the incorporation of [³H]-uridine in RNA and incorporation of [¹⁴C]-aminoacids in proteins. While fetal calf serum and estradiol stimulate the incorporation of both uridine and afno acids, progesterone did not show any effect. Estradiol treated vaginal cells show typical fcroridges (indicative of keratinization of cells) in contrast to estradiol deprived cells, which show microvilli on cell surface when examined in scanning electron microscope.     

A New Liquid Medium for Trypanosoma (Schizotrypanum) cruzi*

A new liquid medium, with fetal calf serum as the sole undefined component, was devised for the cultivation of Trypanosoma cruzi. The need for the serum is ascribed to its mitogenic proteins, which stimulate division of, and the uptake of [³H]thymidine by the parasites. In the new medium, T. cruzi has a cycle culminating in the appearance of up to 90% metacyclic forms in the stationary phase. This cycle is repeated on each serial transfer.     

Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro

Three approaches were investigated for improvement of in vitro maturation (IVM), in vitro fertilization (IVF), and early embryonic development in cattle. These were: 1) Selection of oocytes, 2) medium supplementation with fetal calf serum (FCS) and cow sera (DO, Dl, D10, and D20 to correspond with estrus, metestrus, diestrus, and proestrus, respectively), and 3) addition of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol-17β (E₂)during maturation. Greater proportions (percentage) of oocytes initially selected for their compact cumulus cells completed IVM and IVF when compared to unselected oocytes (P < .05). Proportions (percentage) of selected oocytes that matured and cleaved after in vitro insemination according to serum type used for IVM were: FCS: 110/175 (62.9%) and 37/110 (33.6%) and DO: 130/145 (89.7%) and 52/130 (40.0%); D1 127/130 (97.7%) and 41/127 (32.3%); D10 95/98 (96.9%) and 35/95 (36.8%); D20:113/116 (97.4%) and 49/113 (43.4%). A higher proportion (P < .05) of embryos resulting from the D20 group reached four- and eight-cell stages. In FCS-supplemented maturation media with no hormones added during maturation (control), results of IVM and IVF were 157/265 (59.2%) and 39/157 (24.8%), respectively. With E₂ (1 μg/ml) and FSH (5 μg/ml), comparable results were 189/215 (87.9%) and 71/189 (37.6%); with E₂ (1 μg/ml) plus LH (10 μml), 280/327 (85.6%) and 111/280 (39.6%). Added hormones improved IVM results (P < .05) and, when FSH or LH was added with E₂, in vitro development to four- and eight-cell stages was markedly enhanced (P < .05). Selection of oocytes, D20 serum, and added E₂ and FSH or LH for IVM improved in vitro development of bovine embryos after IVF.     

Changes in the sulfation extent of membrane-associated proteoglycans produced by sertoli cells in culture

Sertoli cells in culture synthesize two different membrane-associated proteoglycans (MA-PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS-PG containing only CS-GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total ³⁵SO₄ incorporation into MA-PG and a shift in the elution profile of each component subjected to ion-exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA-PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE-Sephacel columns at higher salt concentration than the MA-PG synthesized by cells cultured in 0% or 1% FCS. Double-labeled experiments showed that the ³⁵SO_4/3H-glucosamine ratio incorporated into MA-PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA-PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA-PG molecules produced by Sertoli cells.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

Effects of serum,its protein and lipid extracts,and commercial serum proteins and lipid on the isolated frog heart

Summary This study investigates the inotropic effects of serum, its protein and lipid extracts, and commercial serum proteins and lipid on the isolated, spontaneously-beating heart and superfused, hypodynamic ventricle of the frog. Serum taken from either man, horse, calf, frog, or rabbit evoked marked positive inotropic responses which were unaffected by cholinergic, serotonergic, and adrenergic receptor antagonists. Dialysed serum (dialisand) and void volume fractions from Sephadex G200–120 columns corresponding to large molecular weight constituents evoked marked positive inotropic responses. When serum was separated into fractions containing either proteins or lipids/lipoproteins by high-density ultracentrifugation or activated charcoal, both extracts evoked marked positive inotropic responses. Commerical serum globulins and serum containing a high proportion of immunoglobulins elicited large increases in contractile force, whereas serum albumin evoked a negative inotropic effect. Serum which was either boiled and/or treated with chymotrypsin to denature proteins also caused a marked increase in isometric twitch tension in the frog heart. Similar inotropic response was obtained with fractions of boiled serum eluted on columns of Sephadex G200–120. These fractions corresponded to molecular weight in the region of 60–70 kDa. However, the inotropic effect of boiled serum was abolished following pretreatment with lipase. Superfusion of frog hearts with commercial cardiolipin resulted in marked dose-dependent increases in contractile force. The results demonstrate the presence of at least two large molecular weight cardioactive principles in serum. These substances are comparable in size to constituents of serum proteins (e.g., globulins and immuno-globulins) and serum lipids/lipoproteins (e.g., cardiolipin) and may serve as physiological regulators of cardiac function.Abbreviations Ca ²⁺ Calcium - Da dalton - IgG immunoglobulins - Na ⁺ Sodium - K ⁺ potassium     

Picogram detection levels of asialofetuin via the carbohydrate moieties using the light addressable potentiometric sensor

Fetal calf serum asialofetuin was assayed in the sandwich format using biotinylated and fluoresceinated ricin toxin (B-RCA and F-RCA). The sandwiched species was captured on a biotin-BSA coated nitrocellulose membrane with streptavidin. Anti-fluorescein antibody-urease conjugate was bound to the complex, and detected and quantitated under microvolume conditions using the light addressable potentiometric sensor. As little as 250 pg of asialofetuin was detectable whereas fetuin gave no response at conditions as high as 32 ng. Using a competitive inhibition assay, we established that the binding constant for the asialofetuin-ricin complex was 3.6×10⁸ m ^–1. This is in good agreement with data published using glycopeptides derived from asialofetuin, and RCA and the ricin agglutinin, RCA₁₂₀.     

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture

We studied the effect of fetal calf serum and serum protein fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine β-globulins reduced this transfer, although to a lesser extent than whole serum. α-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A γ-globulin fraction failed to exhibit any effect on the uptake of [¹⁴C]phosphatidylcholine by the cells.All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles preincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine to parenchymal cells.These observations were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of the liposomal surface membrane as a result of the binding of certain serum proteins. On the other     

Serum is a rich source of ligands for the scavenger receptor of hepatic sinusoidal endothelial cells

The present study was prompted by findings in our laboratory showing that serum effectively inhibits scavenger receptor (SR)-mediated endocytosis in hepatic sinusoidal endothelial cells (SEC). Experiments with SEC in vitro showed that the presence of 20% human serum inhibited endocytosis of SR ligands, ¹²⁵I-formaldehyde treated bovine serum albumin (FSA) and ¹²⁵I-nidogen, by 30 and 50%, respectively, whereas pre-heated foetal bovine serum (10%) inhibited endocytosis of ¹²⁵I-FSA by as much as 56%. Human, bovine and rat serum had similar inhibitory effect on endocytosis in SEC. Fractionation of foetal bovine and human serum on anion exchange chromatography demonstrated that the inhibitory principle co-purified with macromolecules of high negative charge. The serum fraction that most effectively inhibited SR-mediated endocytosis of ¹²⁵I-FSA did not affect mannose receptor-mediated endocytosis of ¹²⁵I-mannan to the same extent. Trap-labelled negatively charged serum fraction administered intravenously to rats was eliminated almost exclusively by liver, with a blood decay of 50% over the first 3 min after injection. Isolation of liver cells showed that the populations of Kupffer cells and SEC contained 39 and 61% of liver radioactivity 30 min after injection of trap-labelled negatively charged fractions prepared from pre-heated (complement inactivated) foetal bovine sera. These findings suggest that the process of serum formation from native blood generates appreciable amounts of macromolecules that compete specifically with the SR for endocytosis in SEC. The inhibitory power of pre-heated serum is particularly great. For this reason pre-heated serum should be used with caution in studies of SR in SEC.     

Membrane abnormalities of Huntington's chorea fibroblasts in culture.

Huntington's Chorea is an autosomal dominant disease of the nervous system. Proliferating fibroblasts of one such case express metabolic and morphological abnormalities in addition to delayed adhesion to plastic substratum when compared to age, sex and passage number matched human fibroblasts when grown in a minimal essential medium supplemented with glycine or serine and the macromolecular fraction of fetal calf serum. The abnormalities expressed by Huntington's Chorea fibroblasts are fully corrected when the fibroblasts are grown in whole non-filtered fetal calf serum or when 10^?3 M glucosamine is added to the culture medium.     

Effects of exogenous ganglioside GM1 on different stages of cell spreading studied by directly quantifying spreading rate

Abstract

We developed novel methods to directly quantify cell spreading rate. By comparing our methods with traditional methods, we found that the enhancement effects of fetal calf serum (FCS) or the inhibitory effects of exogenous ganglioside GM1 occurred at different stages of cell spreading. GM1 mainly influenced the early and late stages of cell spreading of HUVECs. In the presence of 0.5% FCS, GM1 significantly impaired the area-based spreading rates (127.4 ± 35.7 μm²/h and 22.2 ± 3.8 μm²/h, respectively) on the early (0–0.5 h) and late (12–24 h) stages of cell spreading compared with the controls (238.1 ± 11.7 μm²/h and 35.4 ± 19.5 μm²/h, respectively), which was confirmed by the data on the GM1-induced changes in average length of actin filaments during cell spreading. The real-time observation and quantification of cold-induced de-spreading of GM1-free or GM1-treated HUVECs further confirmed that GM1 can influence cell de-spreading process having inhibitory (0–10 min) or enhancement (10–20 min or 40–50 min) effects on different stages. The methods can be recruited for investigating effects of other reagents on different stages of cell spreading.     

Uptake of fetal proteins by Trypanosoma cruzi immunofluorescence and ultrastructural studies

Isolated proteins from fetal calf serum needed for trypanosomal growth were labelled with I¹²⁵, colloidal gold and fluorochrome tagged specific antibodies. The proteins were localized in the membranes and cytoplasm of parasites cutured in vitro in a defined liquid medium.     

Affinity chromatography of estrogen- and progesterone-binding proteins of human uterus

The purification of estrogen- and progesterone-binding proteins of human uterus by employing affinity resins coupled with steroid-bovine serum albumin conjugates, led to the isolation of preparations with estrogen- and progesterone-binding sites havingK _d values in the range of 0.96 to 1.20 × 10^-9 M. These were different from theK _d values of 10^-10 M and 10^-8 M obtained for two types of binding sites present in the crude cytosolic and nuclear fractions. The purified proteins sedimented on sucrose gradient withS values in the range of 3.6–4.4. The cytosolic and nuclear estrogen- and progesterone-binding proteins, thus purified, showed differences in specificity of binding to the hormone. While the cytoplasmic proteins were more specific in their binding to estradiol or progesterone, the nuclear proteins bound Cortisol with equal or moderate affnity. These results demonstrate the presence of distinct physiological forms of estrogen- and progesterone-binding proteins in the cytoplasm and nucleus, thus pointing to the importance of both these compartments in hormone action.     

Biochemical properties of canine serum ferritin: iron content and nonbinding to concanavalin A

A sandwich enzyme-linked immunosorbent assay using H-subunit-rich canine heart ferritin as a standard has been developed for measuring canine serum ferritin which is H-subunit-rich. Serum ferritin concentrations in 51 normal dogs ranged from 143 to 1766 ng ml^–1, with a mean value of 479±286 (SD) ng ml^–1. Serum ferritin iron concentrations as determined by an immunoprecipitation technique ranged from 30.4 to 115.9 ng ml^–1 in 15 normal dogs with serum ferritin protein levels of 298 to 959 ng ml^–1. There was a significant linear correlation between the serum ferritin iron and protein levels (r=0.9441, P<0.001), and the mean iron/protein ratio of serum ferritin was 0.112±0.017. When canine sera were incubated with concanavalin A-Sepharose 4B, we observed the apparent binding of serum ferritin to concanavalin A. However, ferritin obtained by heat-treating the sera at pH 4.8 to remove the ferritin-binding proteins did not bind to the lectin. These results suggest that canine serum ferritin contains a considerable amount of iron but no concanavalin A-binding G subunit present in human serum ferritin.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a K_M for putrescine of 2.58×10⁻⁶ m and a V_max of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a K_I of 1.8×10⁻⁸ m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The V_max for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the K_M for putrescine, 50.3×10⁻⁶ m. The K₁ of the ABS putrescine oxidase for aminoguanidine was 41×10⁻⁶ m. On the basis of both the K_M and K_I values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.     

Cultured hepatoma cells for the study of enzyme regulation: Induction of ornithine decarboxylase by insulin and asparagine

Summary The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combintion, of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10⁻⁵ M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present. This work was supported in part by Grants CA-07175, CA-22484, and CA-17334 from the National Cancer Institute. D. P. G. is a Predoctoral Fellow at the Food Research Institute, supported by a fellowship from the Monsanto Fund and by NIH Grant R01-AI 15693 to Prof. Michael W. Pariza, Food Research Institute, University of Wisconsin, Madison.     

Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells

Background aimsAs angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells.MethodsThe usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs).ResultsExpression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities.ConclusionsThe use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs.     

Differential Staining of Degenerating Fascicles in Peripheral Nerves

A study has been made on the possibility of replacing leucofuchsin by colored basic fuchsin for the histochemical demonstration of aldehydes. Several tissues from mammals and various pertinent fixatives were used. Aldehydes were freed from carbohydrates by oxidation and from thymonucleic acid by hydrolysis.

It was found that the colored form and not necessarily the leucoform of basic fuchsin can be used histochemically in demonstrating aldehydes. The technic used is as follows: (1) Treat with 1.0–0.5% H₅IO₆ (or in 1% KIO₄ in M/1 H₂SO₄) for 5 to 10 min. and wash thoroughly. For thymonucleic acid hydrolize with N HCl 5 min. at room temperature, 10 min. at 60°C. and 5 min. at room temperature. (2) Stain for 2–3 min. with 0.05% basic fuchsin in 5% ethanol, 3% phenol. (3). Transfer immediately to 1 or 2 changes of 1% sodium bisulphite or potassium metabisulphite in 0.1–0.2 N H₂SO₄ for a total of 5 min. (4) Rinse with water and treat with M H₂SO₄ in 95% ethanol for 3–5 min. 6. Wash thoroughly in water and dehydrate, clear, and mount. For glycogen and mucin the following counterstaining solution is recommended: orange G, 0.25 g.; light green SFY, 0.10 g.; phosphotungstic acid 0.50 g.; 50% ethanol, 100 ml.; glacial acetic acid, 0.25 ml.     

Serum stimulation of sodium transport in human fibroblasts containing low and high levels of intracellular sodium

Summary The relationships between intracellular sodium content, sodium transport and serum effects were investigated in human fibroblasts. In the cells with low intracellular sodium (Na _iL ^/+ ;0.04 mol sodium/mg protein) serum stimulated the sodium-potassium pump as measured by ouabain-sensitive sodium efflux and rubidium influx and also exerted a transstimulation of ouabain-insensitive sodium transport resulting in net influx. In cells with high intracellular sodium (Na _iH ^/+ ;0.42 mol sodium/mg protein) all aspects of sodium transport were increased compared to Na _iL ^/+ cells. In these cells serum caused no change in sodium-potassium pump activity but significantly increased the ouabain-insensitive sodium fluxes resulting in net efflux. In Na _iL ^/+ cells, serum promoted net sodium influx through an amiloride-sensitive pathway that was undetectable in the basal state. In Na _iH ^/+ cells the serum-stimulated net efflux was amiloride sensitive but this pathway also contributed to a major portion of sodium transport in the basal state. This study demonstrated that sodium-potassium pump activity is directed by the supply of internal sodium and that serum can increase this supply by promoting net influx, and that serum-induced sodium transport can be modified by intracellular sodium content.     

Characterization of a serum factor that decreases albumin mRNA in cultured hepatocytes

Summary When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes, and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate orN-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography gave a molecular weight 60 000 to 70 000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with³⁵S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free or serum-supplemented media, and no difference between detergent-treated FBS and control FBS. Therefore, FBS contains a factor that causes a significant decrease in steady state levels of mRNA for albumin and other mRNAs of tissue specific function, but under these conditions albumin mRNA levels are not paralleled by synthesis of albumin or other proteins.