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1.
Extraction of an erythrotropin-like factor from bovine serum albumin (cohn fraction V) 总被引:2,自引:0,他引:2
L. Fernando Congote 《In vitro cellular & developmental biology. Plant》1987,23(5):361-366
Summary Different batches of commercially available bovine serum albumin (Cohn fraction V) were tested in a serum-free medium for
their ability to stimulate thymidine incorporation in erythroid cells of fetal bovine liver. All preparations stimulated thymidine
incorporation. Crystallized, charcoal-treated, or fatty acid-free albumin had substantially lower thymidine incorporation-stimulating
activities than the crude preparations. The albumin preparations also had a synergistic effect with respect to erythropoietin
on erythroid cells from rat liver, a typical property of erythrotropins. One gram of one of the batches of Cohn fraction V
was fractionated by reversed-phase high performance liquid chromatography (HPLC). The fraction with thymidine incorporation-stimulating
activity had a similar elution position as erythrotropin isolated from fetal bovine serum. Further purification using reversed-phase
HPLC in the presence of trifluoroacetic acid and heptafluorobutyric acid and gel permeation HPLC resulted, in the isolation
of a factor that is very similar to fetal bovine serum erythrotropin. It has practically the same specific activity as the
purified fetal peptide in the rat liver bioassay. These results suggest that many of the beneficial effects of the albumin
preparations added as supplement of serum-free tissue culture media may be due to the presence of erythrotropin-like factors.
The work was supported by grants MT-6072 and ME-9031 from the Medical Research Council of Canada. The author is a Chercheur-Boursier
of the Fonds de la Recherche en Santé du Quebec. 相似文献
2.
A heparin-binding erythroid cell stimulating factor from fetal bovine serum has the N-terminal sequence of insulin-like growth factor II 总被引:1,自引:0,他引:1
Q G Li R Blacher F Esch L F Congote 《Biochemical and biophysical research communications》1990,166(2):557-561
An 8 kd heparin-binding peptide which stimulates thymidine incorporation in cultures of fetal calf liver erythroid cells was isolated from fetal bovine serum by affinity chromatography on Heparin-Sepharose, ion exchange chromatography, gel filtration and reversed-phase HPLC. The N-terminal sequence of the isolated peptide was identical to the N-terminal sequence of bovine erythrotropin or insulin-like growth factor II (IGF II). The potential heparin-binding site of IGF II is probably situated in the arginine-rich C-peptide region. The affinities of human recombinant IGF I and II were compared with those of apolipoprotein H (a plasma heparin-binding protein) and bovine insulin in a heparin-affinity column. The retention times were in the order: Apolipoprotein H greater than hrIGF II greater than hrIGF I greater than insulin (no retention). This unusual property of IGF II suggests that it may be captured in the extracellular matrix in a similar way to fibroblast growth factor, interleukin 3 or granulocyte/macrophage colony-stimulating factor. 相似文献
3.
L F Congote 《Biochemical and biophysical research communications》1985,126(2):653-659
Serum erythrotropin (ET) was isolated from fetal bovine serum. Partial sequence analysis of the N-terminal portion of the peptide indicated that the first 20 amino acids were practically identical to those found in human insulin-like growth factor II (IGF II). The effect of IGF II on [3H] thymidine incorporation in cell cultures of fetal bovine liver was similar to the effect of ET. Both factors acted synergistically with erythropoietin but not with platelet derived growth factor. The stimulation of thymidine incorporation by ET and IGF II on cell cultures of fetal liver erythroid cells was at least 15 times higher than their effects on cultures of fetal calf intestine, lung and kidney cells. 相似文献
4.
K V Honn J A Singley W Chavin 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1975,149(2):344-347
Chemical and endocrine parameters were investigated in commercially available fetal bovine sera intended for use as culture media supplements. A high degree of serum variability was present both within and between suppliers in all major categories investigated. It is suggested that caution be employed in the interpretation of results from experiments utilizing serum supplements without specific quantitation of possible interfering or modulating factors. 相似文献
5.
L F Congote 《Biochemical and biophysical research communications》1985,133(2):404-409
An erythrotropin-like peptide (ELP) and bovine serum erythrotropin (ET) coeluted after more than four chromatographic steps. ELP was separated from ET on gel permeation high pressure liquid chromatography due to its large molecular weight. The effect of ELP on the stimulation of [3H] Thymidine incorporation in cell cultures of fetal bovine liver was much lower than the effect of ET. The N-terminal amino acid sequence of ELP indicated that it has a group of amino acids identical to portions of the LDL-receptor and the hypothetical proteins BHLF1 and BOLF1 deducted from the DNA sequence of the Epstein-Barr virus. These results are consistent with the idea that cell surface proteins have structural homologies with secreted proteins or some growth factor-related peptides. 相似文献
6.
Clouet-Dumas A Le Bizec B Le Pape M Maume D Monteau F Andre F 《The Journal of steroid biochemistry and molecular biology》2000,74(1-2):57-62
Usually performed to investigate biotransformations of xenobiotics, in vitro liver models could become useful tools for the synthesis of not commercially available compounds. In this study, bovine hepatocyte cultures were used to biosynthesise, on the laboratory scale, one major metabolite of methyltestosterone: 6beta-hydroxymethyltestosterone. After incubation of bovine hepatocytes with methyltestosterone for 24 h, culture medium was removed and stored at -20 degrees C until analysis. The sample was extracted and purified on a reversed-phase HPLC system. The metabolite of interest was then analysed in LC-MS and GC-MS for structural identification. The purity and the isomery of the 6 and 17 positions were confirmed by NMR analyses. This first success in producing purified 6beta-hydroxymethyltestosterone from bovine hepatocyte cultures allowed us to consider that in vitro liver models could be reliable tools for standard biosynthesis. 相似文献
7.
Johan B. Ubbink W. J. Hayward Vermaak Siegbert Bissbort 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1991,565(1-2)
A rapid, isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of total homocysteine levels in human serum. Prior to reversed-phase HPLC analysis, the serum thiols were derivatized with SBD-F (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate), a thiol-specific fluorogenic probe which is commercially available. Retention of SBD-homocysteine was sensitive to pH, and a mobile phase pH of 2.1 ensured baseline separation of serum thiols within 6 min. The method is simple, sensitive, reproducible (between-run coefficient of variation of 6.6%) and very suitable for routine determination of serum homocysteine levels in a clinical pathology laboratory. 相似文献
8.
Lynn L. Stoll Arthur A. Spector 《In vitro cellular & developmental biology. Plant》1984,20(9):732-738
Summary The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs
widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2)
and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms: number of double bonds). Swine
serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid
(22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different
serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse
serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid
18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however,
growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when
the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of
22:6. The fact that the cellular fattyacid composition is likely to change should be taken into account when changes are contemplated
in the serum used to grow established cell lines.
These studies were supported by Arteriosclerosis Specialized Center of Research Grant HL 14,230 from the National Heart, Lung,
and Blood Institute, National Institutes of Health. 相似文献
9.
Four commercially available fortified sera were compared to fetal bovine serum (FBS) with regard to their ability to maintain or increase the sensitivity of the Buffalo green monkey (BGM) kidney cell line to viral infection. Nine virus strains and five wastewater samples were used. Fortified sera were comparable to FBS for the enumeration of some viruses by the plaque method and for the detection of virus in wastewater by the most-probable-number assay. 相似文献
10.
Four commercially available fortified sera were compared to fetal bovine serum (FBS) with regard to their ability to maintain or increase the sensitivity of the Buffalo green monkey (BGM) kidney cell line to viral infection. Nine virus strains and five wastewater samples were used. Fortified sera were comparable to FBS for the enumeration of some viruses by the plaque method and for the detection of virus in wastewater by the most-probable-number assay. 相似文献
11.
Summary Two commercially available serum replacements developed for use in the culture of hybridoma and other mammalian cells were
tested for their suitability as replacements for fetal bovine serum in insect cell culture medium. CPSR-1 and CPSR-3 both
supported growth of the insect cell line IPLB-SF-21AE. CPSR-3 supported adequate growth, but cells in medium supplemented
with CPSR-1 grew much slower and achieved only about half the final cell density of either FBS or CPSR-3 supplemented medium.
This work was supported in part by grant 187159 from the Juvenile Diabetes Foundation and BRSG RR05876 from the National Institutes
of Health, Bethesda, MD. 相似文献
12.
Mikael Knip Pentti Lautala Hans K. Akerblom Kauko Kouvalainen Julio M. Martin 《Life sciences》1983,33(23):2311-2319
An activity that enhances insulin release from perifused rat pancreatic islets has recently been isolated from human serum fractions (molecular weight 1,000–5,000 daltons). To characterize this activity we have studied the insulin-releasing effect of serum subfractions from obese and non-obese children obtained by reversed-phase high-performance liquid chromatography (HPLC). The serum insulin-releasing activity eluted in the HPLC system at 12–13 minutes, which corresponded to the retention time of the tridecapeptide insulin-glucagon liberin isolated from bovine hypothalamus. Insulin-releasing activity was found in serum subfractions from both obese and normal-weight children. The relative insulin-releasing potency of the active subfractions was higher than that of the original total serum fractions, indicating the presence of some substance(s) which inhibit insulin secretion in the total serum fractions. Oral glucose loading increased the relative insulin-releasing activity in the HPLC subfractions from obese children. This study suggests that the insulin secretagogue in human serum might be identical to hypothalamic insulin-glucagon liberin as these substances behave similarly on reversed-phase HPLC and have parallel insulin-releasing properties. 相似文献
13.
N-terminal peptides of bovine ribonuclease (RNase) of 20, 13 and 7 amino acid residues were isolated by reversed-phase high-performance liquid chromatography (HPLC). Antibodies were raised in mice against these peptides coupled to bovine serum albumin (BSA). It was shown that antibodies against the peptides reacted with the intact protein and that the immune response decreased with decreasing size of peptide. In order to obtain a satisfactory reaction with the intact protein, the peptide immunogen should be longer than 7 amino acids. 相似文献
14.
A rapid and sensitive HPLC-based method for quantitating isoaspartate levels in peptides and proteins is described. The analyte is incubated for 40 min with S-adenosyl-l-methionine and the commercially available enzyme protein l-isoaspartyl methyltransferase. Methylation of isoaspartyl sites results in stoichiometric production of S-adenosyl-l-homocysteine that is separated from the other components of the reaction by reversed-phase HPLC and quantitated online by absorbance at 260 nm. This method can accurately detect 5 pmol or less of isoaspartate and works with tryptic digests as well as intact proteins. Using a commercially available isoaspartyl peptide, the relationship between isoaspartate levels and S-adenosyl-l-homocysteine production was found to be linear and stoichiometric over a range of 5-250 pmol. Compared to methods that measure [(3)H]methanol production after methylation with S-adenosyl-l-[methyl-(3)H]methionine, the HPLC method is safer, faster, less expensive, and equally sensitive. 相似文献
15.
Insulin-stimulating peptide from a tryptic digest of bovine serum albumin: purification and characterization 总被引:1,自引:0,他引:1
A procedure was established for isolation of a low molecular weight polypeptide with insulin-stimulating activity in apparent homogeneity from a tryptic digest of bovine serum albumin on a semipreparative scale. Purification of this insulin-stimulating peptide (ISP) was monitored by an adipose-explant assay in which stimulation of fatty acid synthesis from glucose by insulin was measured. The polypeptide was purified by a combination of DEAE-cellulose column chromatography, gel filtration on Bio-Gel P-10, hydrophobic chromatography on a semipreparative C18 reversed-phase HPLC column, and ion exchange chromatography on an SP-5PW HPLC column. The primary structure of ISP was deduced. ISP is a two-chain polypeptide consisting of 71 amino acid residues, and corresponds essentially to residues 115-143 and 144-184 (185) of bovine serum albumin connected to each other by a disulfide bridge. But comparison of the sequence of ISP with that of the relevant regions of bovine serum albumin determined by Brown indicated the presence of one tyrosine insertion between residues 155 and 156 of albumin. Therefore, the molecular weight of ISP was calculated to be 8,496. 相似文献
16.
A sandwich ELISA for bovine serum in viral vaccines 总被引:4,自引:0,他引:4
A double antibody sandwich ELISA for the detection of bovine serum in viral vaccines was developed and standardized with commercially available reagents. The detection limit by ELISA was 0.5 ng ml-1. ELISA was found to be 50-400 times more sensitive than the currently used assays. It was concluded that ELISA is a specific, sensitive and reproducible method for the determination of residual amounts of bovine serum in viral vaccines. 相似文献
17.
Eggs were flushed from the oviducts of slaughtered gilts that had been inseminated after synchronization of estrus and ovulation with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Of 347 eggs collected at a recovery rate of 73.8%, 41.8% had not cleaved by 32 h after expected ovulation time. Of those cleaved, 86.9% were at the two-cell stage.Two-cell embryos were cultured in Dulbecco's medium containing either no protein or 20% of one of the following: lyophilized bovine serum albumin, lyophilized fetal calf serum or heat-inactivated serum from slaughter heifers, rabbits or barrows. Dulbecco's medium without protein did not support further development of embryos. Addition of heat-inactivated blood serum from slaughter heifers, rabbits or barrows resulted in development rates similar to those obtained by using commercially available products. Optimal embryonic development rates of 54.5% were obtained with addition of heat-inactivated bovine serum. Of the two-cell embryos only three (2.1%) developed past the four-cell stage in these culture media. 相似文献
18.
Ylva Blixt Anders Valeur Einar Everitt 《In vitro cellular & developmental biology. Plant》1990,26(7):691-700
Summary Plasma membranes isolated from HeLa cells cultivated in suspension cultures supplemented with 3.5% fetal bovine serum or 2%
of the commercially available serum substitute Ultroser G contained the same amounts of protein, cholesterol, and phosphate
on a cellular basis. Minor differences in the plasma membrane fatty acid composition were seen, with the most pronounced alteration
observed for palmitic acid, which amounted to 27 and 20% in fetal bovine serum- and Ultroser G-supplemented cells, respectively.
Plasma membranes from cells growth with Ultroser G contained almost twice as much phosphatidylethanolamine and displayed two
thirds of the phosphatidylcholine content, compared to plasma membranes obtained from fetal bovine serum supplemented cells.
The former membranes also showed a 3 times higher specific [3H]acetate labeling of cholesterol, indicating a higherde novo synthesis of cholesterol. Both quantitative and qualitative alterations were revealed among the plasma membrane polypeptides
when these were subjected to immuno- and lectin blottings. Fluorescence anisotropy measurements at different temperatures
produced similar results irrespective of the growth medium supplement when the plasma membrane specific probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene
was used on intact cells. However, the average cellular rigidity was higher for Ultroser G supplemented cells, determined
with 1,6-diphenyl-1,3,5-hexatriene as a probe.
This investigation was supported by grants from the Swedish Natural Science Research Council, Anders Otto Sw?rds Stiftelse,
Stockholm, Crafoordska Stiftelsen, Lund and Kungl. Fysiografiska S?llskapet, Lund. 相似文献
19.
Kitagawa Hiroshi; Tsutsumi Kae; Ujikawa Miho; Goto Fumitaka; Tamura Jun-ichi; Neumann Klaus W.; Ogawa Tomoya; Sugahara Kazuyuki 《Glycobiology》1997,7(4):531-537
The relationship between sulfation and polymerization in chondroitinsulfate (CS) biosynthesis has been poorly understood. In thisstudy, we investigated the specificity of bovine serum UDP-GalNAc:CS ß-GalNAc transferase responsible for chain elongationusing structurally defined acceptor substrates. They consistedof tetra- and hexasaccharide-serines that were chemically synthesizedand various regular oligosaccharides with a GlcA residue atthe nonreducing terminus, prepared from chondroitin and CS usingtesticular hyaluronidase. The enzyme preparation was obtainedfrom fetal bovine serum by means of heparin-Sepharose affinitychromatography. The preparation did not contain the 相似文献
20.
In vitro cultivation of Dipetalonema viteae third-stage larvae: evaluation of culture media, serum, and other supplements 总被引:1,自引:0,他引:1
Third-stage larvae of Dipetalonema viteae obtained from the tick vector developed to the fourth stage in several cell-free culture systems. Survival and development of larvae in a number of commercially available cell culture media, supplemented with serum and other defined and undefined components, were compared. All cultures were gassed with 5% carbon dioxide in nitrogen. Best survival, growth and development were obtained in stationary cultures containing 1:1 (v/v) mixtures of NCTC 135, either RPMI 1640 or Iscove's Modified Dulbecco's Medium, and a supplement of 20% non-heat-inactivated fetal bovine serum. The importance of the medium composition and physical environment of the culture system, for the survival, growth and development of D. viteae was demonstrated. 相似文献