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1.
ICHIRO KIMURA TAKAYUKI HASEGAWA EIJIRO OZAWA 《Development, growth & differentiation》1982,24(4):369-380
Myotrophic activity of highly purified chick transferrins (Tfs) to chick primary myogenic cells has been studied in a culture medium containing horse serum. Iron-binding to Tfs is indispensable for the activity. The removal of iron from Tfs gives rise to a complete loss of the activity and it is restored by iron-rebinding depending on the amount of bound iron. This result, combined with other physicochemical and immunological data, strongly, confirms that the myotrophic activity is exerted by the Tfs themselves, not by a contaminating material(s). It has been found that culture medium containing horse Tf which seems inadequate for the study of the biological effects of Tfs is, however, suitable for studies on chick Tfs, since horse Tf is inactive in promoting chick myogenesis. Terminal sialic acid residues are unrelated to myotrophic activity since Tfs with different numbers of residues (0, 1, and 2 moles/Tf molecule) are comprable in their activities. The mechanism of Tf action on cells and contradictions among previous papers as to the requirement of Tf for cell growth have been discussed from the viewpoint of an iron-donor with class-specificity. 相似文献
2.
As an extension of previous studies, we reexamined the developmental change in trophic activity of chicken serum on chicken myogenic cells in vitro and attempted to elucidate it on the basis of possible changes in serum transferrin (Tf), the myotrophic activity of which depends both on its concentration and on the level of its iron-saturation. The myotrophic activity was found to be low until the second week in ovo , then to increase rather abruptly to a plateau at about the time of hatching, and then to decrease to the adult level. Determination of the concentration and level of iron-saturation of serum Tf suggested that the change in myotrophic activity was mainly caused by these two parameters, though another factor(s) may also be involved. 相似文献
3.
Class specificity of transferrin as a muscle trophic factor 总被引:2,自引:0,他引:2
The specificity of transferrin (Tf) in its exertion of a growth-promoting effect on myogenic cells was examined using serum Tfs from chick, dove, goose, turkey, bovine, horse, rabbit, rat, and swine and primary myogenic cells from chick, duck, quail, rabbit, and rat, and rat L6 cells. Avian Tfs were effective on avian cells but not on mammalian cells, while mammalian Tfs were effective on mammalian cells but not on avian cells. Dove and bovine Tfs were exceptional in that they were effective on some class-heterologous cells at higher concentrations and less so or completely ineffective on some class-homologous cells. Despite these exceptions, however, the relationship between Tfs and cells can be summarized as a class specificity. To exert the growth-promoting effect, it is prerequisite for Tf to bind its specific receptor on the cell surface. Using quail and L6 cells, we found that the binding of 125I-labeled chick and rat Tfs to the respective receptors of quail and L6 myoblasts was competitively inhibited by other kinds of effective Tfs, but not by ineffective ones. We conclude that the class specificity in myotrophic activity of Tf is due to the affinity between Tf and Tf receptor. 相似文献
4.
Necessity of transferrin for RNA synthesis in chick myotubes 总被引:3,自引:0,他引:3
Chick transferrin (Tf) is essential not only for growth and differentiation but also for the maintenance of chick myotubes in culture. Its removal from the culture medium gives rise to degeneration of the myotubes. The analysis of this process revealed that the removal resulted in decrease in total and messenger RNA content in the myotubes; this was mainly due to a decrease in RNA synthesis. Activity of in vitro RNA synthesis in isolated nuclei from myotubes cultured without Tf was lower than the activity in nuclei from myotubes cultured with Tf and increased with the addition of FeCl3. Although RNA degradation in myotubes was also enhanced following Tf removal, the degree was small. The synthesis of most proteins was reduced. In contrast to this, a few new proteins of unknown nature were synthesised in myotubes cultured in Tf-free medium. The role of Fe ion carried into the cells by Tf in promoting myogenic cell growth and differentiation and in preventing the myotubes from degeneration can be explained, at least in part, on the basis of its effect on RNA synthesis. Since we have found that Fe is required for activation of RNA polymerase purified from embryonic muscles (Shoji and Ozawa, 1985b), these effects may be ascribed to this activating effect. 相似文献
5.
Chick Embryo Extract, Muscle Trophic Factor and Chick and Horse Sera as Environments for Chick Myogenic Cell Growth 总被引:1,自引:0,他引:1
YASUKO HAGIWARA ICHIRO KIMURA EIJIRO OZAWA 《Development, growth & differentiation》1981,23(3):249-254
Chick myogenic cells grew in a medium composed of Eagle's minimum essential medium (MEM), horse serum (HS), and one of the essential factors needed for myogenic cell growth (EFMG), that is, chick embryo extract (EE), chick serum (CS), or the muscle trophic factor (MTF). But they did not grow in the absence of the EFMG. In the absence of HS, they scarcely grew in a medium composed of MEM, and EE or MTF. They grew in a medium composed of MEM and CS; they grew much better in a medium composed of MEM, CS, and HS.
In the presence of one of the EFMG, the optimal HS concentration for growth varied depending on its lot. At higher HS concentrations, growth was suppressed. Further, it was suggested that an inhibitory substance(s) for myogenic cell growth was present in HS. The inhibitory effects can usually be minimized by diluting the serum with an artificial medium. 相似文献
In the presence of one of the EFMG, the optimal HS concentration for growth varied depending on its lot. At higher HS concentrations, growth was suppressed. Further, it was suggested that an inhibitory substance(s) for myogenic cell growth was present in HS. The inhibitory effects can usually be minimized by diluting the serum with an artificial medium. 相似文献
6.
Iron supports myogenic cell differentiation to the same degree as does iron-bound transferrin 总被引:2,自引:0,他引:2
T. Hasegawa, K. Saito, I. Kimura, and E. Ozawa (1981, Proc. Jopan Acad. B 57, 206-210) have shown that Fe ion can promote myogenic cell growth as Fe-bound transferrin. In the present work, the effects of these substances in supporting myogenic cell differentiation were examined. The hallmarks of differentiation adopted were appearance of structural and regulatory proteins, myofibrils, sarcoplasmic reticulum, and Ca-activated activities of myosin B and phosphorylase kinase; isoform transition of creatine kinase; and acquisition of cell membrane excitability and contractility following electrical stimulation of myotubes. The degree of differentiation of myotubes cultured in the presence of Fe ion was almost the same as that of myotubes cultured in the presence of Fe-bound transferrin. These facts suggest that transferrin protein molecules do not play a primary role in differentiation. Further, it has also been shown that myotubes acquire excitation-contraction and metabolism coupling qualitatively similar to that of adult muscle fiber. 相似文献
7.
Samples of homozygous bovine serum transferrins have been prepared and their purity has been ascertained by immunological techniques and electrophoretic analysis in SDS. Measurements of carbohydrate composition show that no significant differences exist among the phenotypic variants AA, D1D1, D2D2, and EE. Chromatography of transferrin AA on DEAE-cellulose separated four subfractions, each of which corresponded well with one band obtained by polyacrylamide gel electrophoresis. Carbohydrate analyses of the individual subfractions did not show significant differences in sialic acid, hexose, or hexosamine contents. After desialylation with neuraminidase, each subfraction was converted to a major band and a minor band on gel electrophoresis. From the relative band positions of the desialylated transferrins, it was concluded that possession of sialyl residues by bovine transferrin is not the primary cause of electrophoretic multiplicity. Rather, sialic acid masks an underlying heterogeneity which most likely resides within the polypeptide chain. Further characterization of this heterogeneity will best be undertaken with the isolated asialotransferrin subfractions.This research was supported by Grants MT-4074 and MA-5554 from the Medical Research Council of Canada and a Senior Fellowship (to M. W. C. H.) from the Ontario Heart Foundation. 相似文献
8.
Aluminium (Al) in the blood is bound to transferrin (Tf), a glycoprotein of about 80kDa that is characterized by its need for a synergistic anion. In this focused review, the binding affinity of Al to Tf is surveyed in the context of our recent studies using on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry (HPLC/HR-ICP-MS). Al in human serum without any in vitro Al-spikes was present in a form bound to the N-lobe site of Tf. The influences of sialic acid in the carbohydrate chain of human serum Tf (hTf) were studied using asialo-hTf, obtained by treatment with sialidase. The binding affinity of Fe was similar between asialo-hTf and native-hTf, while that of Al for asialo-hTf was larger than that for native-hTf, especially in the presence of oxalate, a synergistic anion. The above findings are discussed in relation to diseases in which the serum concentrations of carbohydrate-deficient Tf and oxalate are augmented. 相似文献
9.
In starch gel electrophoresis of horse sera each transferrin variant is formed by a strong anodal band and a weaker cathodal band. An 'atypical' variant, Tf C, has two zones of about equal intensity. Family data show that Tf C is genetically controlled by an allele Tf C at the Tf locus. Frequencies of transferrin alleles in various horse breeds are also presented.
After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephad Summary ex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of bands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid. 相似文献
After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephad Summary ex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of bands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid. 相似文献
10.
Transferrin stimulates iron absorption, exocytosis, and secretion in cultured intestinal cells 总被引:1,自引:0,他引:1
The cellularmechanism by which basolateral transferrin (Tf) produces an increase inapical-to-basolateral Fe flux in Caco-2 cells was analyzed. After apulse of 59Fe from the apicalmedium, three types of basolateral59Fe efflux were found: a59Fe efflux that was independentof the presence of Tf in the basolateral medium, a59Fe efflux in which59Fe left the cell bound to Tf,and a Tf-dependent 59Fe efflux inwhich 59Fe came off the cell notbound to Tf. Furthermore, addition of Tf to the basolateral mediumdoubled the exocytosis rate of Tf and increased the secretion ofapolipoprotein A, a basolateral secretion marker. Both apotransferrinand Fe-containing Tf produced similar increases in59Fe efflux, Tf exocytosis, andapolipoprotein A secretion. The Ca2+ channel inhibitor SKF-96365inhibited both the Tf-mediated increase in transepithelial Fe transportand the secretion of apolipoprotein A. Thus the activation oftransepithelial Fe transport by Tf seems to be mediated byCa2+ entry into the cells. 相似文献
11.
We have shown that triiodothyronine-dependent GH1 rat pituitary cell growth in serum-free defined culture required apotransferrin (apoTf) (D. A. Sirbasku, et al., Biochemistry 30, 295-304, 7466-7477, 1991). These studies were done in "low-Fe" medium without Fe(III)/Fe(II) salts. Nonetheless, significant concentrations of iron may have been contributed by other components, making this medium unsuitable for study of the differential effects of apoTf and diferric transferrin (2Fe.Tf). Measuring residual iron in culture medium has been troublesome because the most sensitive method (i.e., atomic absorption) detected levels only in excess of 10 ng/ml and did not distinguish between the forms of iron present. To estimate the Fe(III) available to bind to apoTf, we developed a more sensitive and specific method. Urea-polyacrylamide gel electrophoresis (PAGE) separates apoTf, the two monoferric transferrins, and 2Fe.Tf. [125I]apoTf was incubated with medium, or components, and the formation of [125I]-2Fe.Tf was monitored by urea-PAGE/autoradiography. By this method, the concentration of Fe(III) in low-Fe medium was estimated at 8.4 to 20 ng/ml and the sources were identified. We next sought to remove the Fe(III). Standard chelators were ineffective or cytotoxic. In contrast, an affinity method with deferoxamine-Sepharose depleted greater than or equal to 90% of the Fe(III). In this medium, apoTf and 2Fe.Tf showed differential effects with GH1 cells and with MCF-7, MTW9/PL2, an MDCK cells. With the methods described here, the effects of apoTf and 2Fe.Tf on growth can be studied separately. 相似文献
12.
Angiotensin converting enzyme in cultured endothelial cells and growth medium. Relationships to enzyme from kidney and plasma 总被引:1,自引:0,他引:1
We have previously reported that cultured cells from swine aorta possess angiotensin converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) and release it into serum-free culture medium. The present work compares enzyme from these two sources, and from swine kidney and serum, with respect to antibody and lectin binding. Purified enzyme from swine kidney, and the activity in swine serum, cultured endothelial cells and culture medium bind similarly to rabbit antibodies prepared against the kidney converting enzyme. Enzyme from each of these sources was allowed to bind to an immobilized lectin (Ricinus communis), which binds to terminal galactose residues of glycoproteins. Increasing concentrations of galactose were used to remove enzyme from the lectin column and the distribution of enzyme activity in the galactose eluates was determined. The elution pattern was similar for kidney and endothelial cell enzyme, and different from the pattern found for both serum and medium enzymes. Neuraminidase treatment of either serum or medium enzyme altered the distribution of activity eluted to that found for endothelial cell or kidney enzymes. The effects of neuraminidase suggest that the difference in lectin binding between cell and medium enzyme reflects differences in the number of terminal sialic acid residues that cover galactose residues. 相似文献
13.
ICHIRO KIMURA 《Development, growth & differentiation》1983,25(6):531-535
Developmental change in microheterogeneity of chicken serum transferrin (Tf) was investigated by polyacrylsmide-gel isoelectric focusing, direct immunofixation, and densitometry. Three main Tf species (Tf-0, Tf-1, and Tf-2, which have 0, 1, and 2 sialic acid residues per molecule, respectively) were resolved and their relative ratios were determined. As development proceeded, a relative increase occurred in the most acidic species (Tf-2) with decreases in the less acidic ones (Tf-0 and Tf-1). 相似文献
14.
Nuevo-Ordoñez Y Montes-Bayón M Blanco González E Sanz-Medel A 《Metallomics : integrated biometal science》2011,3(12):1297-1303
Serum transferrin (Tf) is an iron binding glycoprotein that plays a central role in the metabolism of this essential metal but it also binds other metal ions. Four main transferrin forms containing different iron binding states can be distinguished in human serum samples: monoferric (C-site or N-site), holotransferrin (with two Fe atoms) and apotransferrin (with no metal). Recently, it has been reported that Tf binds also Ti even more tightly than does Fe, in artificially Ti(iv) spiked solutions. However, very limited work has been done on the Ti binding to Tf at physiological concentrations in patients carrying intramedullary Ti nails. Here we report the chemical association of Ti to Tf "in vivo" under different chromatographic conditions by elemental mass spectrometry using double focusing inductively coupled plasma (DF-ICP-MS) as detector. For the separation of the Ti/Fe-Tf forms different gradient conditions have been explored. The observed results reveal that human serum Ti (from patients carrying intramedullary Ti nails) is uniquely associated to the N-lobe of Tf. The investigation of the influence of sialic acid in the carbohydrate chain of human serum Tf, studied by incubating the protein with neuraminidase (sialidase) to obtain the monosialilated species, revealed that the binding affinity of Ti was similar for monosialo-Tf and for native-Tf and occurs in the N-lobe. These results suggest that the species Fe(C)Ti(N)-TF might provide a route for Ti entry into cells via the transferrin receptors after the release of the metal from its implants. 相似文献
15.
Summary The aim of this study was to compare quantitatively the capacity to transcytose (i.e. to uptake and release) transferrin (Tf) with the pinocytic activity of suspended adult rat hepatocytes. An oligodisperse preparation of131I-polyvinylpyrrolidone (PVP;M
r 36000) was used to measure the inward and outward aspects of the pinocytic process in separate experiments. Cell association of rat125I-Tf was measured at Tf concentrations approaching physiological, where59Fe uptake obeyed first-order kinetics. Release studies with both PVP and Tf were carried out under conditions which minimized the probability ofde novo endocytosis of a molecule already released. Sets of experimental points representing cell-associated radioactivities were converted into continuous algebraic functions by fitting with two-term (release studies) or three-term (uptake studies) exponential equations. Transport of PVP and Tf through the cells was computed from these equations by deconvolution. This analysis showed that, under the present experimental conditions, the fractional transcytosis rates of Tf and PVP by hepatocytes were in the ratio of I:0.77. These values imply that, in the physiological range of Tf concentrations, about 75% of the Fe taken up by hepatocytes may be due to a pinocytic mechanism (fluid-phase or mixed). Inclusion of chloroquine (1 mM) in the suspending medium, both in uptake and release experiments, resulted in more PVP and Tf passing through the cells, while Fe uptake was reduced. It is suggested that the base probably exerted its enhancing effect on transcytosis by shunting the subcellular transport of PVP and Tf to the outward leg through a shorter circuit.Abbreviations BSA
bovine serum albumin
- HBSS
Hank's balanced salt solution
- Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- MEM
minimal essential medium
- PVP
polyvinylpyrrolidone
- Tf
transferrin 相似文献
16.
Abdurrahim Kocyigit Ozcan Erel Mehmet S. Gurel Senel Avci Necmeddim Aktepe 《Biological trace element research》1998,65(3):271-281
The aim of this study was to measure the alterations in serum selenium (Se), copper (Cu), zinc (Zn), and iron (Fe) concentrations
and their carrier proteins, ceruloplasmin (Cp), transferrin (Tf) albumin, and related antioxidant enzyme activities, erythrocyte
Cu-Zn Superoxide dismutase (Cu-Zn SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities in patients with cutaneous
leishmaniasis (CL). Erythrocyte Cu-Zn SOD activities, serum Cu concentrations, and Cp levels were found to be significantly
higher in the patients group than those of controls. However, GSH-Px and CAT activities and Se, Zn, Fe, and Tf levels were
lower in patients than in the control subjects. There were positive important correlation’s between Cu-Zn SOD and Cp, Cu-Zn
SOD and Cu, Cp and Cu, GSH-Px and Se, and Fe and CAT in the patients group. Our results showed that serum essential trace
elements Se, Zn, Cu, and Fe concentrations and their related enzymes Cu-Zn SOD, GSH-Px, and CAT activities change in CL patients.
The changes may be a part of defense strategies of organism and are induced by the hormonelike substances. 相似文献
17.
Kiyoshi Ohkawa Koji Takada Naoko Takizawa Takashi Hatano Yutaka Tsukada Makoto Matsuda 《FEBS letters》1990,270(1-2):19-23
Human ovarian clear cell carcinoma cell line (transferrin (Tf)-non-producer), HAC 2, cells were adapted to grow in chemically defined synthetic medium when the cells were cultured with medium containing 10 μg/ml of insulin at least for 6 months. They synthesized and secreted constantly the 80 kDa protein immunologically similar to human serum Tf(15 ± 12 ng/ml/107 cells/3 days). By sensitive lectin-affinity electrophoresis followed by antibody-affinity blotting technique, a concanavalin A weakly bound or unbound, lentil lectin, a strongly reactive abnormal band, which was rarely found in human serum Tf, was detectable in the Tf synthesized by HAC 2 cells (HACTf). These findings suggest that the HACTf may act as one of the autocrine growth factors and that this heterogeneity of HACTf for lectin affinity is ascribed to differences in the carbohydrate moiety of the Tf. 相似文献
18.
Serum samples from 412 Spanish wild rabbits were analysed by starch and polyacrylamide gel electrophoresis. Three different transferrin (Tf) phenotypes (A, AB and B) were observed by both methods. The occurrence of two codominant alleles (TfA and TfB with frequencies of 0.89 and 0.11 respectively) at an autosomal locus (Tf) was supported by the population data on genetic equilibrium. Electrophoretic mobility differences between the Tf variants A and B could not be explained by differences in sialic acid or iron contents. Each of the two Tf variants were shown to have two sialic acid residues by neuraminidase treatment. These variants had similar affinities for iron, and iron binding did not lead to the conversion of one variant into the other. 相似文献
19.
Farnaud S Amini M Rapisarda C Cammack R Bui T Drake A Evans RW Suryo Rahmanto Y Richardson DR 《The international journal of biochemistry & cell biology》2008,40(12):2739-2745
Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site. 相似文献
20.
Transferrins (Tfs) are glycoproteins with carbohydrate chains in the C-lobe. Carbohydrate-deficient Tfs (CDTs) with fewer sialic acids increased in several diseases. In this study, the affinity of metals (Al and Fe) to Tfs was compared between native- and asialo-Tf by on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry, to clarify whether the presence of sialic acids influences the metal binding. Fe added as Fe-citrate in the presence of bicarbonate preferred the N-lobe site and the binding affinity was similar between native- and asialo-Tfs. Al-citrate added at Al/Tf = 1 also preferred the N-lobe site, while the binding affinity was higher to asialo-Tf than to native-Tf. In Al-oxalate addition, the affinity to the N-lobe site of both Tfs increased further. In the absence of bicarbonate, Al-oxalate showed a preference for the C-lobe site in native-Tf and comparable affinity to both lobes in asialo-Tf. In asialo-Tf, Al2-Tf was the largest peak even at Al/Tf = 1. Thus, the lack of sialic acid in glycans and the presence of oxalate enhanced the binding affinity of Al to Tf. Therefore, it was suggested that the binding affinity of Al in patients with CDTs may be enhanced. 相似文献