Bovine Serum Transferrin Research Articles

Regulation of deoxyribonucleic acid synthesis in proliferating and differentiating trophoblast cells: involvement of transferrin, transforming growth factor-beta, and tyrosine kinases

This report investigates the regulation of DNA synthesis in trophoblast stem cells and differentiating trophoblast cells. Experiments in this study were performed on the Rcho-1 trophoblast cell line, which was established from a transplantable rat choriocarcinoma. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate along the trophoblast giant cell pathway. DNA synthesis in quiescent trophoblast stem cells (maintained in serum-free medium) was stimulated by fetal bovine serum and donor horse serum or transferrin to a level approximately 30- and 10-fold above the basal level, respectively. Transferrin and horse serum were ineffective at maintaining trophoblast cell proliferation. In contrast, serum-starved differentiating trophoblast cells synthesize DNA at maximal levels and could not be further stimulated by the addition of exogenous factors. Fetal bovine serum-stimulated proliferation was effectively inhibited by transforming growth factor-beta 1. Experiments with the tyrosine kinase inhibitor genistein implicate tyrosine kinase involvement in the regulation of DNA synthesis and proliferation in trophoblast stem cells and DNA synthesis in differentiating trophoblast cells. Proliferating and differentiating trophoblast cells differ in their levels of tyrosine kinase activities and express unique tyrosine-phosphorylated proteins. In summary, DNA synthesis and proliferation in trophoblast stem cells are under extrinsic control, whereas DNA synthesis in differentiating trophoblast cells is under intrinsic control. Both mechanisms require tyrosine kinase activity, but the nature of the tyrosine kinase pathways in each process appears to be distinct.

Analytical method optimization for protein determination by fast high-performance liquid chromatography

A method based on fast HPLC allowing the rapid and efficient determination of proteins in mammalian cell cultures is proposed, involving the use of two chromatographic modes, RP-HPLC and high-performance liquid affinity chromatography. These two sequential chromatographic analyses separate the proteins most often used as supplements in serum-free media such as bovine serum albumin, insulin and transferrin, and also monoclonal antibodies secreted by hybridoma in cultures. Rapidity, reliability and flexibility are the main characteristics of the method. The monitoring of proteins in the course of a discontinuous hybridoma culture is presented.

Identification of bovine serum transferrin phenotypes by polyacrylamide gel isoelectric focusing (PAGIEF).

Each of four common homozygous bovine serum transferrins Tf A, Tf D1, Tf D2, and Tf E gave only two main bands in polyacrylamide gel isoelectric focusing (PAGIEF). This is considered to be due to the co-migration of the main components 2a and 3a with the main components 2b and 3b, respectively, in PAGIEF. Ten phenotypes, which are controlled by four alleles TfA, TfD1, TfD2, and TfE, were distinguishable from each other in PAGIEF.

Development of Cell Cultures Derived from Lake Trout Liver and Kidney in a Hormone-Supplemented, Serum-Reduced Medium

Abstract Primary cultures of liver cells and kidney cells of lake trout Salvelinus namaycush were initiated in a hormone-supplemented, serum-reduced medium in plastic dishes. Cells plated on fibronectin-coated plastic attached with high efficiency (87–93%), in contrast to cells cultured on uncoated or polymer-coated plastic (< 8–52%). Primary cultures were obtained from young lake trout by trypsin-EDTA dissociation at 10–15°C for 40 min; the primary cultures were maintained in a growth medium (either medium 199 or Leibovitz's L-15 medium) supplemented with 2–3% fetal bovine serum, epidermal growth factor, bovine serum albumin, cholera toxin, insulin, transferrin, and selenium. Lake trout liver and kidney cultures currently contain both epithelioid and fibroblastic cells. The cells, cultivated at 18–20°C from primary culture, have been subcultured 32 times over a period of 6 months.

Simple method for the preparation of antigen emulsions for immunization

We have developed a rapid, safe, and reliable method to prepare emulsions of water-soluble antigens in an adjuvant oil phase for immunization purposes. The method, based on well established emulsification principles, employs a three-way ‘T’ connector to which three disposable syringes are attached. The system allows the stepwise addition of small volumes of the water phase, into the oil phase. We have compared the time required for emulsification, the rate of antigen release from the emulsion into a physiological phase, and the immunogenic properties of bovine serum albumin and transferrin contained in emulsions made by the new stepwise addition method, with those made by the widely used double-hubbed needle method. We report a significantly shorter ( P < 0.001) and a more reproducible emulsification time for the stepwise addition method (6.1 ± 2.1 min; mean ± SD) than for the double-hubbed needle method (41.1 ± 28.0 min). The stepwise addition method always yielded water-in-oil emulsions, while the double-hubbed needle method failed, about 20% of the time, to produce a water-in-oil emulsion after 120 min of mixing. Since the stepwise addition method employs a connector with a larger inner diameter (1.75 mm) than the one required for the double-hubbed needle method (0.84 mm); the pressure required for the former is markedly reduced compared with that required for the latter, thus making the new method safer and less labor-intensive. The rate of antigen release from the emulsions was significantly slower when the stepwise addition method was employed ( P < 0.01). There were no differences in viscosity and stability in the emulsions prepared by the two methods. The ability of antigen-containing emulsions to elicit an immune response was found to be identical by the two methods; no significant differences were found in antibody titers as determined by enzyme-linked immunosorbent assays. These characteristics make the stepwise addition system the method of choice.

Hapten‐tagged plasma proteins as immunocytochemical probes for the study of vascular permeability

Bovine serum albumin and transferrin were covalently coupled with fluorescein isothiocyanate and digoxigenin, respectively, and intravenously co-injected in equal amounts in mouse. The derivation of the two proteins induces minor alterations of their physicochemical properties as well as of their physiological functions. The two tracers were revealed within vascular and extravascular compartments of diaphragm by quantitative postembedding immunocytochemistry, using antibodies against each of the haptens in conjunction with the protein AG-gold complexes. The influence of different fixatives and embedding protocols on the immunodetectability of the hapten-tagged proteins was assessed. Both resist reasonably well to osmication and embedding in Epon. None of the haptens reacted with the heterologous antibody. At 30 minutes after injection, the tracers were detected in blood plasma, interstitium, and endothelial plasmalemmal vesicles. The presence of both proteins within the interendothelial clefts was inconspicuous. The ratios between the labeling densities found over endothelium, interstitial space, and vascular lumen were similar for both tracers. This suggests that the endothelium of mouse diaphragm capillaries might exhibit comparable permeabilities towards serum albumin and transferrin which are similar in size and charge. The study shows that hapten-tagged polypeptides are close to the corresponding native macromolecules, and represent interesting tools for the morphological study of dynamic processes such as transcytosis.

Role of cyclic anp in the regulation of serotomin N-acetyltransferase in retina

A rabbit polyclonal antiserum was raised against bovine serum transferrin (TF) and used to construct a homologous radioimmunoassay which measures bovine TF in the range 0.16–4.00 μg/ml. TF is a transport protein which is believed to supply iron to developing germ cells which reside beyond the blood-testis barrier. The assay was used to monitor TF secretion by cultures of Sertoli cells which were isolated from 3–4-month-old Hereford calf testes and maintained in culture for up to four days. The cells were treated with a range of hormones and growth factors in serum-free medium to determine which factors regulate the secretion of TF.Basal levels of TF secretion were low (0.16–0.26 μg/ml); however, major increases (8-fold; P < 0.001) in the secretion of TF were induced by bovine follicle-stimulating hormone (FSH; half-maximal response (ED50)=310 ngUSDA-bFSH-Bl/ml) and by the more purified ovine FSH (ED50=5.5 ngNIADDK-oFSH-16/ml). The nucleotide analogue 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) also evoked a 9-fold increase in TF secretion (ED50=0.71 mmol/l), indicating that the FSH response was mediated via the activation of adenylate cyclase. Significant increases in TF secretion of a lesser magnitude were also induced by epidermal growth factor (EGF; 121% at 500 ng/ml), insulin (348% at 20 IU/ml), and retinol (531% at 500 ng/ml) and all three agents augmented the TF response to FSH. In contrast, testosterone (500 nmol/l) showed only a 55% stimulation of TF secretion, and all other steroids (5α-DHT, oestradiol-17β, cortisol, progesterone) and protein hormones tested: bovine luteinizing hormone (⩽ 50 ng NIAMMD-bLH-4/ml) prolactin (⩽ 100 ng NIAMMD-bPRL-6/ml) and glucagon (⩽ 2 μg/ml) were without significant effect. These experiments show that both hormones (FSH, insulin) and growth factors (EGF, retinol) regulate TF secretion in the bovine Sertoli cell.

High accuracy molecular weight determination and variation characterization of proteins up to 80 ku by ionspray mass spectrometry

A quadrupole mass spectrometer with an ionspray interface was used to measure the molecular weight (MW) of proteins up to 80,000 u. With the improvements in instrument calibration by a statistical averaging method and in data analysis by a gaussian curve-fitting method, precision of MW determination as high as 12 ppm was achieved with equine myoglobin (MW 16,950 ± 0.2 u). Exact MW determination of three components in cerato-ulmin revealed that the two minor ones had lost amino acid residues Ser and Ser-Asp, respectively, from the major component (MW 7618.4 ± 0.2 u). MW classification of eight components in the Fab fragment of a monoclonal antibody revealed that one set of four had MW ∼ 47,540 u and the other ∼ 47,640 u. The MW difference of 100.2 ± 0.6 u between fragment 1 and 2, attributed to inhomogeneous cleavage at the Fab C-terminus, was probably due to one additional Thr in 1. The MW of bovine serum albumin (BSA) was found to be 66,431.5 ± 1.3 u, ∼ 164 u higher than the calculated sequence MW, most probably because of the incorrectness in the previously reported BSA amino acid sequence. The MW of human serum transferrin (79,556.8 ± 1.7 u) was shown to be 4414 u higher than the sequence MW, pointing to a glycosylation of 22.7 sugar units in this protein. The greater complexity in bovine serum transferrin (MW 78,030.5 ± 1.8 and 78,326 ± 3.3 u for the two major components) was correlated with the heterogeneity in the glycosylation.

In vitro cultivation of adult Litomosoides carinii: evaluation of basic culture media, gas phases and supplements

Adult Litomosoides carinii, recovered from cotton rats (Sigmodon hispidus) 4-5 months post-infection (p.i.), were cultivated in vitro with emphasis on investigations into the development of intra-uterine embryonic stages. Baseline values for the embryonic status of female worms were established immediately after recovery from the hosts. In such females, on average, 16% of the intra-uterine stages were fully formed microfilariae while the remainder belonged to the early embryonic classes that were characterized. For the evaluation of culture success, apart from survival of worms in vitro, the rate of microfilariae development (mf rate) served as a major parameter. Of the five basic culture media RPMI 1640, F12, L15, NCTC 135, and IMDM, tested without supplementation, RPMI 1640 yielded by far the best results (survival = 14 days; mf rate = 41%), and was therefore chosen as the routine medium. In comparison with 5% CO2 in nitrogen, a gas phase of 5% CO2 in air was superior, although the resulting oxygen tension of 138 mmHg in the medium was not physiological. Addition of 10% newborn or foetal bovine serum to the medium in some cases distinctly influenced results. Effects of different batches of sera varied from 'filaricidal' to 'very promoting'. Co-cultivation of worms and Sigmodon cells, or rhesus monkey LLCMK2 cells, only marginally improved results. Of the serum substitutes Ultroser G, BMS, and Clex, the latter had a moderately promoting effect. The protein supplements bovine serum albumin, transferrin and haemoglobin significantly improved results, and could replace certain batches of serum. Supplementation with the haemin moiety alone was less effective than with haemoglobin. The anti-oxidants glutathione plus ascorbic acid proved beneficial in combination with a serum supplement only. Results from other experiments with multiple supplementation also suggest that various supplements may act only in a synergistic manner. The longest average time that adult L. carinii survived in vitro was 3-4 weeks. The highest mf rate was 78%, which indicated that all normal embryonic stages present in the uteri of a female at the start of culture completed their development to microfilariae, however, oogenesis ceased in vitro. The parameters for embryonic development employed proved to be highly sensitive for the judgment of various culture conditions.

Clonal growth and self-renewal of human myeloid leukemia cell lines (BRM and DD) in serum-free semi-solid culture

Primary and secondary colony formation of two new human myeloid leukemia cell lines (BRM and DD) were studied in serum-free semisolid cultures. The results indicate that bovine serum albumin and transferrin were essential for clonal growth in chemically defined medium. Insulin contributed only moderately beneficial effects. Initial cell density was also a major modulator of plating efficiency. Positive cooperation between the leukemia cells was shown by using autologous conditioned media. This is the first serum-free culture method that allows self-renewal of human myeloid leukemia cell lines in terms of secondary colony formation in methylcellulose cultures.

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, beta-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated.

Synthesis of Transferrin and Transferrin mRNA in Bovine Sertoli Cells in Culture and in Vivo: Sequence of Partial cDNA Clone for Bovine Transferrin1

Techniques were developed for generating enriched cultures of bovine Sertoli cells and indifferent supporting cells (immature Sertoli cells). The [35S]methionine and [35S]sulfate-labeled proteins secreted by cultured cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The electrophoretic pattern of the major Sertoli cell-secreted proteins was distinct from that of the major proteins secreted by cultured peritubular cells (the predominant contaminating cell type). Five major polypeptides ranging in molecular mass from 22 kDa to 77 kDa were resolved by 2D-PAGE in reducing conditions and were assigned numbers for reference purposes. Polypeptides 1 and 2 appeared to be analogous to two rat Sertoli cell-secreted proteins, sulfated glycoprotein-1 and sulfated glycoprotein-2, because of similar molecular mass, isoelectric point, subunit composition, sulfation, and sialation characteristics. Transferrin was detected in conditioned medium by immunoprecipitation using an antibody to bovine serum transferrin. Cultured Sertoli cells isolated from prepubertal bulls secreted higher levels of transferrin than did cells isolated from infant bulls. An 850 bp cDNA corresponding to the 3' portion of bovine transferrin mRNA was cloned and sequenced. Transferrin message was shown to be present in testicular tissue isolated from infant and prepubertal bulls and it increased as bulls matured. Levels of testicular transferrin mRNA were subsequently shown to correlate with daily sperm production in yearling beef bulls.

Diffusional kinetics of monoclonal antibody, transferrin and bovine serum albumin on Sephacryl S-200 HR gel

The adsorption and diffusional kinetics of three macromolecules, namely bovine serum albumin (BSA), transferrin and monoclonal antibody (McAb), on Sephacryl S-200 high resolution gel were studied by the pulse-response chromatographic technique in a column of diameter 1.6 cm and length 8.5 cm. Experimental results showed that the voidage ε and axial mixing (2D L /V) in the packed column were 0.432 and 0.05 cm based on the response to a pulse input of blue dextran 2 000 000 and D 2O solution respectively. The equilibrium constant K and pore diffusivity D p were 0.23 and (0.60 – 1.02) × 10 −7 cm 2 s −1 for BSA (molecular weight, 67 000) 0.17 and (0.62 – 1.0) × 10 −7 cm 2 s −1 for transferrin (molecular weight, 95 000) and 0.095 and (0.53 – 0.85) × 10 −7 cm 2 s −1 for McAb (molecular weight, 150 000 IgG). A van't Hoff plot of ln K vs. 1/T gave a straight line with zero slope and hence zero heat of sorption for all three components. This implies that no adsorption occurs between the molecules and the packing, and that the partition of solute in the bulk solution and pore solution is entirely controlled by diffusional forces.

Ultrathin-layer isoelectric focusing for bovine serum transferrin phenotyping

We successfully removed sialyl residues from the Tf molecule by neuraminidase treatment and succeeded in phenotyping cattle serum using ultrathin-layer isoelectric focusing

Ultrathin-layer isoelectric focusing for bovine serum transferrin phenotyping

Ultrathin-layer isoelectric focusing for bovine serum transferrin phenotyping

Regulation of transferrin secretion in Sertoli cells of the calf testis

A rabbit polyclonal antiserum was raised against bovine serum transferrin (TF) and used to construct a homologous radioimmunoassay which measures bovine TF in the range 0.16–4.00 μg/ml. TF is a transport protein which is believed to supply iron to developing germ cells which reside beyond the blood-testis barrier. The assay was used to monitor TF secretion by cultures of Sertoli cells which were isolated from 3–4-month-old Hereford calf testes and maintained in culture for up to four days. The cells were treated with a range of hormones and growth factors in serum-free medium to determine which factors regulate the secretion of TF. Basal levels of TF secretion were low (0.16–0.26 μg/ml); however, major increases (8-fold; P < 0.001) in the secretion of TF were induced by bovine follicle-stimulating hormone (FSH; half-maximal response (ED 50)=310 ng USDA- bFSH- Bl/ ml) and by the more purified ovine FSH (ED 50=5.5 ng NIADDK- oFSH-16/ ml). The nucleotide analogue 8-bromo-cyclic adenosine monophosphate (8Br-cAMP) also evoked a 9-fold increase in TF secretion (ED 50=0.71 mmol/l), indicating that the FSH response was mediated via the activation of adenylate cyclase. Significant increases in TF secretion of a lesser magnitude were also induced by epidermal growth factor (EGF; 121% at 500 ng/ml), insulin (348% at 20 IU/ml), and retinol (531% at 500 ng/ml) and all three agents augmented the TF response to FSH. In contrast, testosterone (500 nmol/l) showed only a 55% stimulation of TF secretion, and all other steroids (5α-DHT, oestradiol-17β, cortisol, progesterone) and protein hormones tested: bovine luteinizing hormone (⩽ 50 ng NIAMMD-bLH-4/ml) prolactin (⩽ 100 ng NIAMMD-bPRL-6/ml) and glucagon (⩽ 2 μg/ml) were without significant effect. These experiments show that both hormones (FSH, insulin) and growth factors (EGF, retinol) regulate TF secretion in the bovine Sertoli cell.

Effects of serum and serum-derived factors on the growth and production of ?-fetoprotein and albumin by human hepatoma cell lines

A serum-free culture system of human hepatoma cell lines (HuH-6 and HuH-7) was used to investigate the activity of bovine serum (BS) and of serum-derived factors on the growth and production of alpha-fetoprotein (AFP) and albumin. At higher concentrations, dialyzed BS was inhibitory to the growth of HuH-6 and caused reduction of the level of AFP production by the cells. AFP and albumin levels in HuH-6 and HuH-7 were reduced or unchanged by fetuin, bovine serum albumin (BSA) and transferrin (TF), although no cytotoxicity was shown by any of them. Commercial preparations of platelet-derived growth factor exhibited cytotoxicity to HuH-6 and HuH-7 and induced a decrease of AFP and albumin levels in a dose-dependent manner. Transforming growth factor (TGF-beta) exhibited no cytotoxicity to HuH-6. AFP levels in HuH-6 were unchanged with 1000 pg/ml TGF-beta, but albumin levels were decreased. TGF-beta at a concentration of 1000 pg/ml was cytotoxic to HuH-7 and AFP levels were a little increased. Albumin levels, however, were unchanged. Following exposure to cycloheximide, AFP and albumin levels in HuH-6 were inhibited.

A growth-promoting factor for human myeloid leukemia cells from horse serum identified as horse serum transferrin

A growth-promoting factor for human myeloid cells was purified to apparent homogeneity from horse serum by a combination of gel filtration, blue Sepharose affinity chromatography, Mono Q anion-exchange chromatography, Mono P chromatofocusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The growth promoter was an iron-bound, single glycopolypeptide chain with a molecular weight of 84,000, an isoelectric point of 5.4 and an amino terminal sequence of Glu-Gln-Thr-Val-Arg-Trp-Cys-Thr-Val-Ser-Asn-His-Glu-Val-Ser-Lys-. According to the results of the amino acid sequence, iron binding ability and physicochemical properties, we identified the growth-promoting factor as horse serum transferrin. It was highly active in promoting the proliferation of a human monocytic leukemia cell line, THP-1, as well as of two other human myeloid cell lines, HL-60 and K-562. It had the same activity in proliferating THP-1 cells as 5% fetal calf serum-supplemented medium. Horse serum transferrin could be substituted for human or bovine serum transferrin.

Isolation and purification of an antibody to 67-KD calcimedin.

The 67-KD calcimedin is a calcium-binding protein isolated from several muscle tissues. The protein shows apparent Mr of 67,000 by SDS-polyacrylamide gel electrophoresis. An antibody has been prepared by immunizing sheep with the protein purified from chicken gizzard smooth muscle. This antibody recognizes 67-KD calcimedin but not calmodulin, bovine serum albumin, transferrin, or brain p68 calelectrin. The presence of 67-KD calcimedin is demonstrated in the smooth muscle cell lines A10 and DDT1MF-2 as well as in primary cultures of chicken breast and heart muscle, by immunoprecipitation and immunofluorescence. The 67-KD calcimedin, being responsive to calcium, may play a role in calcium-mediated cell regulation. This report identifies several cells that may be useful for further delineation of the cellular role of 67-KD calcimedin.

Human AML colony growth in serum-free culture

Here we report experiments dealing with the development of a fully serum-free culture system for AML cell proliferation. A number of compounds were tested for the requirements of AML colony formation and DNA synthesis in vitro in an attempt to replace serum with an artificial mixture. The results indicate that bovine serum albumin (BSA, 15 mg/ml), cholesterol (7.8 μg/ml), transferrin (7.7 × 10 −6 M) and insulin (1 μg/ml) were essential for AML cell proliferation. Linoleic acid, 2-mercaptoethanol and selenite contributed only moderately beneficial effects. This mixture obviated the need for adding exogenous fetal calf or horse serum to the cultures and permitted equivalent and frequently superior AML colony formation and DNA synthesis. This is the first serum-free culture method for human AML that may allow for in-vitro studies of the growth regulation by recombinant growth factors under exactly defined and standardized conditions.