An erythropoietic stimulating factor similar to erythropoietin released by macrophages after treatment with silica

An erythropoietic stimulating factor (ESF) can be detected in the supernatant from fetal liver and adult bone marrow and spleen cells when preincubated with the macrophage-specific cytotoxic agent, silica. Stimulation is observed in 12-day fetal liver CFU-E cultures in the absence of added erythropoietin (Ep). The concentration of ESF in the supernatant added to CFU-E cultures is dependent on the preincubated cell dose and the volume added. The stimulating activity is abolished when mice are hypertransfused and increased above normal values when mice are bled. A concentrated silica-treated spleen supernatant was able to stimulate erythropoiesis in the polycythemic mouse bioassay. It is concluded that the ESF is similar, if not identical, to Ep.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

In vitro assembly of dogfish brain tubulin and the induction of coiled ribbon polymers by calcium

It was previously shown that BHK21 cells were arrested in the G1 phase of the cell cycle when cultured in medium lacking serine. In this study the effect of serine limitation on protein synthesis was examined. Shifting cells from medium supplemented with 10% fetal calf serum to medium supplemented with 10% dialyzed serum resulted in a 50% reduction in the rate of protein synthesis. The reduced rate was attained within 4–10 min after shift-down and was restored completely within 5–15 min after shift-up to 10% dialyzed serum plus 0.05 mM serine, the same approximate concentration of serine present in 10% fetal calf serum. Exogenous serine appears to be incorporated into protein from a precursor pool which is functionally compartmentalized inasmuch as incorporation of serine into protein became linear within 10 min after the addition of label while the specific activity of serine in the acid soluble fraction did not attain a constant value during 60 min of labeling. The serine: leucine ratio in total cellular protein was determined from cells cultured in ten percent dialyzed serum plus 0.05 mM serine by amino acid analysis and was compared with the ratio of [³H]serine and [¹⁴C]leucine incorporated into protein. The results indicated that 50–60% of the serine utilized for protein synthesis under these conditions was derived from the medium while the other 40–50% was generated within the cell.     

Separation of cells by velocity sedimentation

A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 10⁷ cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r²/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.     

Vasopressin stimulates sulfate incorporation into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture

The effects of lysine vasopressin (1–100 ng/ml) on the 24 h incorporation of [³⁵SO₄⁻] into proteoglycans synthesized by fetal rat chondrocytes in monolayer culture has been investigated. The hormone enhances sulfate incorporation into proteoglycans released in the medium and those associated with the cell layer. This enhancement was independent of cell density or stimulation of cell division by the hormone or calf serum. These observations provide evidence that the hormone stimulation of sulfate incorporation is not directly linked to hormone stimulation of cell division.     

Culture shock. Selective uptake and rapid release of a novel serum protein by endothelial cells in vitro

A novel protein has been purified from fetal calf serum and from serum-free bovine aortic endothelial cell conditioned culture medium. This protein consists of a single polypeptide chain of reduced Mr 70,000 (70K protein) and was separated from bovine serum albumin and other proteins by ion-exchange chromatography and immunoabsorption on Sepharose-coupled anti-70K protein antiserum. The 70K protein was shown to be structurally and immunologically distinct from bovine serum albumin, alpha-fetoprotein, and vitronectin by one- and two-dimensional peptide mapping, amino acid analysis, and enzyme-linked immunosorbent assay and/or immunoblotting. The 70K protein was located in endothelial cell cytoplasmic granules of irregular size and distribution. Metabolic radiolabeling studies showed that the 70K protein was not a biosynthetic product of these cells; its cytoplasmic location was due to a selective uptake from the fetal calf serum in which the cells were initially grown. After subconfluent cultures of endothelial cells were shifted to serum-free medium, nearly 80% of the total 70K protein that was measurable in the medium was released between 0 and 20 min. Moreover, sparse, rapidly proliferating cells released approximately 18-fold more 70K protein within 2 min as compared to dense, nonproliferating cultures. The concentration of 70K protein in fetal calf serum was estimated to be 400-600 micrograms/ml. Proliferating bovine aortic endothelial cells, 24 h after plating at an intermediate density, released approximately 250 pg of 70K protein/cell within the first 20 min after exposure to serum-free conditions. The data provide evidence for a novel protein in serum which is selectively internalized by endothelial cells in vitro and which in turn is released rapidly under conditions such as osmotic imbalance due to serum removal, or during periods of cellular proliferation, conditions which we term "culture shock."     

Identification of light and dark hypertrophic chondrocytes in mouse and rat chondrocyte pellet cultures

Hypertrophic “light” and “dark” chondrocytes have been reported as morphologically distinct cell types in growth cartilage during endochondral ossification in many species, but functional differences between the two cell types have not been described. The aim of the current study was to develop a pellet culture system using chondrocytes isolated from epiphyseal cartilage of neonatal mice and rats, for the study of functional differences between these two cell types. Hypertrophic chondrocytes resembling those described in vivo were observed by light and electron microscopy in sections of pellets treated with triiodothyronine, 1% fetal calf or mouse serum, 10% fetal calf serum or 1.7 MPa centrifugal pressure at day 14, and in pellets cultured with insulin or 0.1% fetal calf or mouse serum at day 21. A mixed population of light and dark chondrocytes was found in all conditions leading to induction of chondrocyte hypertrophy. This rodent culture system allows the differentiation of light and dark chondrocytes under various conditions in vitro and will be useful for future studies on tissue engineering and mechanisms of chondrocyte hypertrophy.     

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.     

Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3′:5′-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([³H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G₁ phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G₁ phase. J. Cell. Biochem. 70:563–572, 1998. © 1998 Wiley-Liss, Inc.     

Differentiation in vitro of mouse embryos to the stage of early somite

Mouse blastocysts continuously differentiate in vitro to the early somite stage with reconstituted rat tail collagen as the substrate for the attachment. In order for this to occur, it appears that two differentiation barriers must be overcome. The first, the formation of egg cylinders from the inner cell mass, can be overcome by incubating embryos in heat-inactivated fetal calf serum. The second, the formation of the early somite from the presomite stage, can be overcome by replacing fetal calf serum with human cord serum.Mouse blastocysts were initially incubated with calf serum in Eagle's minimum essential medium. After shedding the zona pellucida, the denuded blastocysts lay flat on the surface of the collagen. Soon thereafter, trophoblastic cells invaded the underlying collagen leaving the rounded inner cell mass protruding from the surface of the collagen. By replacing calf serum in the medium with fetal calf serum the inner cell mass differentiated into endoderm and ectoderm to form an egg cylinder.The egg cylinder rapidly became elongated and formed extraembryonic and embryonic regions. However, the embryonic region shrank from this point on in the fetal calf serum, and the resulting yolk sac formation did not contain the embryo proper. When fetal calf serum was replaced with human cord serum at the end of the egg cylinder stage (equivalent to embryos of about 7.5 days gestation) neural tissue, cardiac chambers, and somites were formed.     

Interactions of ganglioside GM1 with human and fetal calf sera. Formation of ganglioside-serum albumin complexes

The interactions of ganglioside G_M1 with human and fetal calf sera were studied, the following main results being obtained: (a) G_M1, upon incubation with both sera gave origin to two G_M1-protein complexes, which also occurred after interaction of G_M1 with the albumin fractions prepared from the same sera. Instead no complex formation occurred using the albumin-free fractions. Therefore G_M1 appeared to specifically bind serum albumin and to form G_M1-albumin complexes. (b) G_M1 binding to serum albumin started at ganglioside concentrations surely micellar (above 10^?6 M), was time and concentration dependent, and resulted in a relevant degree of G_M1 complexation (up to 80% of total G_M1 in human serum and up to 18% in fetal calf serum). (c) the binding kinetics appeared, in both serum and the correspondent albumin fraction, to be biphasic: in the first phase, occurring till about 2 · 10^?4 M G_M1, the ratio between bound and total G_M1 increased linearly with increasing G_M1 concentration; in the second phase, occurring above 2 · 10^?4 M, the ratio remained practically constant. After these findings it should be expected that G_M1, when present in serum containing systems, forms complexes with albumin. This should be appropriately considered when studying the effects of exogeneous G_M1 in in vivo and in vitro (tissue cultures) systems.     

Temperature-dependent permeability of large unilamellar liposomes

The temperature-dependent drug leakage from liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (4:1, by weight) was studied. Experiments were performed in Hepes buffer and 50% fetal calf serum. Large unilamellar liposomes were formed by the reverse phase evaporation process and extruded through a series of polycarbonate membranes with pore sizes of 0.4, 0.2, 0.1 and 0.08 μm. The release of the water soluble radioisotopes cytosine 1-β-D-[³H]arabinofuranoside and [³H]inulin from the aqueous compartment of these liposomes was measured as a function of time and temperature. Both radioisotopes were released at temperatures near 42°C, the solid-to-liquid-crystalline phase transition temperature of these lipids. The percent drug release decreased as the size of the liposomes was reduced. This effect was more pronounced in Hepes buffer than serum. The release of both radioisotopes was greatest at 40°C in Hepes buffer and at 43°C in 50% fetal calf serum. In addition, the rate of drug release was much faster in serum than in buffer. These results suggest that different drug release processes are occurring in buffer and in serum.     

Vitronectin is the major serum protein essential for NGF-mediated neurite outgrowth from PC12 cells.

The rat pheochromocytoma cell line PC12 can be induced to differentiate in response to nerve growth factor (NGF) in the presence of 1% fetal calf serum (FCS). Using a novel assay procedure we have developed a purification protocol which has allowed the isolation of the protein in serum responsible for neurite outgrowth after NGF treatment. FCS has been fractionated using four chromatographic procedures and in each case the peak of biological activity copurified with vitronectin. We have concluded, therefore, that vitronectin is the protein present in FCS which can mediate NGF-dependent neurite outgrowth in PC12 cells. Vitronectin and fibronectin from FCS have been chromatographically separated and only the former is capable of inducing neurite outgrowth. We have also shown that vitronectin utilizes the RGD amino acid sequence in binding to the surface of PC12s.     

Spermatogenic cell differentiation in vitro

Culture conditions that support the in vitro development of many spermatogenic stages from the frog Xenopus laevis are described. Spermatogenic cells were dissociated with collagenase and preelongation stages aseptically isolated by density gradient centrifugation in Metrizamide. The cells were then cultured in modified forms of defined nutrient oocyte medium (DNOM). The development of spermatogenic cells was affected significantly by changes in fetal calf serum concentration, cell density, energy sources, and NaCl concentration. Optimum in vitro spermatid development was obtained when spermatogenic cells were cultured at relatively high densities (3–7 × l0⁷ cells/25 cm²) in DNOM modified to contain 10% heat-inactivated, dialyzed fetal calf serum, 2 mM 1-glutamine, 0.1 % glucose, 15 mM HEPES buffer (pH 7.4), and 38.3–48.3 mM NaCl. These culture conditions also supported the differentiation of preelongation spermatids and spermatocytes isolated by density-gradient centrifugation in Metrizamide and subsequent unit gravity sedimentation in gradients of bovine serum albumin. Approximately 95 % of such isolated spermatids and spermatocytes continued differentiating in vitro for 14 days at in vivo rates. Phase-contrast and electron microscopy of the cultured cells demonstrated that in vitro differentiation was morphologically normal between the leptotene and elongate spermatid stages. Autoradiographic studies of preleptotene development demonstrated that spermatogonia proliferated and preleptotene spermatocytes developed to zygotene in 12-day cultures. The results suggest that many spermatogenic stages in Xenopus can develop independent of Sertoli cells, and demonstrate that spermatogenic cell cultures can now be used for in vitro studies of spermatogenesis.     

Insect cell culture media for cultivation of new world Leishmania

Monophasic insect cell culture media were evaluated for suitability in the propagation of strains of Leishmania brasiliensis, L. mexicana and L. enriettii. Amastigotes were inoculated into Grace's, Schneider's and Mitsuhashi-Maramorosch's Media containing antibiotics and varying concentrations of fetal calf serum. After 4–7 days incubation at 27°C, promastigote yields exceeded 1 × 10⁸/rr.l in some instances in contrast to yields of approximately 2 × 10⁷/ml in blood-based media. Optimal concentrations of fetal calf serum were determined to be 15% or more for most of the strains tested.     

LOSS OF IRON FROM MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF [55FE]FERRITIN AND [55FE]FERRITIN RABBIT ANTIFERRITIN COMPLEXES

Mouse peritoneal macrophages in culture for 24 h were exposed to horse [⁵⁵Fe]ferritin and rabbit antihorse [⁵⁵Fe]ferritin antibody complex and the amount of ⁵⁵Fe in the medium was assayed up to 2 days after the pulse uptake. Cell survival was assayed by photographing the same areas of the tissue culture Petri dish on successive days and by counting cell numbers per unit area. In experiments in which quantitative assay for cell death is negligible, about 10–20% of the iron ingested by pinocytosis or phagocytosis is released to iron-free medium containing either freshly dialyzed or deironized newborn calf serum (10%). Over the 2-day postpulse period, iron loss is linear. This loss of iron to the medium is significantly reduced by adding iron-saturated newborn calf serum in the postpulse recovery period. A significant portion of the iron released to the medium is bound to transferrin. When human serum is used in the tissue culture system, similar quantities (10–25%) of the ingested iron are lost to the medium 2 days after the pulse.     

Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor.

An in vitro colony formation assay for the evaluation of in vivo brain tumor therapy has been developed. When plated, disaggregated cells derived from solid tumors proliferated to form relatively homogeneous colonies after a latency period of 2 to 6 days. Increasing concentrations of fetal calf serum enhanced colony-forming efficiency (CFE) with a plateau between 7 and 16%. Supplementation with either irradiated feeder cells (10(3) to 10(5) cells per dish), or medium conditioned by 1 to 3 days of in vitro incubation with the same cell line, doubled the CFE. The density of tumor cells (untreated or previously treated with chemotherapeutic agents) did not affect the CFE when a minimum of 10(4) total cells (tumor plus feeder) were plated. Therefore, in this system the optimal experimental conditions for evaluating chemotherapy and radiotherapy require incubation of disaggregated tumor cells for 12 days in medium containing 10% of fetal calf serum and enough feeder cells to provide a minimum of 10(4) cells per dish. The CFE for untreated tumors was 18 +/- 10% (+/-S.D.), demonstrating that there is significant biological variation. The assay appeared sensitive, with reproducible results, when applied to individual chemically treated tumors. An estimate of the percentage of clonogenic cells affected by in vivo chemotherapy may be obtained by comparing the CFE of cells from treated and untreated tumors. This assay can measure up to a 5 log(10) cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.     

Switch in the synthesis from IgM to IgG in the human lymphoblastoid cell line RPMI-6410t as affected by human sera

The switch from IgM to IgG in lymphoblastoid cell line RPMI-6410t was induced by human sera. The factor inducing the switch was found in the human placental serum and in the serum of peripheral blood of healthy donors. The switch investigated is induced both in the initial line 6410t and in some IgM+ sublines derived from it. With the help of the cloning method some IgG+ sublines were developed with different IgG-synthesis levels from 6410t line and its IgM+ sublines after inducing the switch in them. Earlier another type of the switch induction from IgM to IgA was observed in the same line and its IgM+-sublines by the factors contained in some batches of fetal calf serum (FCSG+). Thus, the homogeneous IgM+ cell population is shown to be able to pass in vitro though two different stages of differentiation inherent to B lymphocytes in vivo.     

A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

An anorectic proteoglycan of membrane origin

The endogenous substance(s) involved in the regulation of food intake has been isolated from serum, urine and feces. In the present study, a similar type of anorexigenic proteoglycan was isolated from human rat erythrocyte membranes and rat liver membranes. Membranes were suspended in 2.0% deoxycholate and allowed to stand at 25 degrees C for 30 min. The suspension was treated with 5% TCA, supernatant was collected, dialyzed and concentrated. TCA-soluble proteins were fractionated on Sephadex G-150. The active second peak fractions were further purified on DEAE-Sephadex A-25. Biologically active substance reduced the appetite in rats significantly when given intraperitoneally. The proteoglycan (50 kDa) consisted of 70-85% carbohydrate. Similar properties of plasma and membrane anorectic substance further indicated its membrane origin. We believe that this anorectic proteoglycan is anchored to cell membranes and released into the blood circulation to regulate the food intake.