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1.
The concentrations of transferrin and albumin in the blood serum and microsomal fraction of the liver and the incorporation of [14C] leucine into the proteins were measured in rats which were fasted while exposed to ambient atmospheric pressure or to a pressure of one-half atmosphere. The rates of protein synthesis were estimated in a relative manner from the ratio of 14C incorporation into the two proteins and in an absolute manner using the liver free 14C and leucine concentrations to measure the specific activity of the precursor pool. Fasting at ambient pressure was accompanied by a decrease in the serum and microsomal concentrations of transferrin but not of albumin and by a marked decrease in the relative and absolute synthesis rates of transferrin. By contrast, fasting at reduced ambient pressure was associated with an increase in the serum transferrin concentration and in the relative and absolute rates of synthesis of the protein. It is concluded that fasting in the rat produces a much greater decrease in the rate of synthesis of transferrin than of albumin and that exposure to reduced ambient pressure stimulates transferrin synthesis but not albumin synthesis. 相似文献
2.
George C. T. Yeoh Paul Vogel John Daly Valerie J. Brighton Alan Light David S. Petterson 《Experimental cell research》1984,151(2)
A group of glycolipid toxins, corynetoxin (CT), isolated from parasitized annual ryegrass, was shown to suppress the synthesis of both albumin and transferrin by cultured fetal rat hepatocytes. Based on [3H]leucine incorporation, inhibition of transferrin synthesis was greater than that of both albumin and total protein synthesis. As a result, the secretion of albumin and transferrin was decreased. The incorporation of [3H]N-AcGlc into cellular glycoproteins was only marginally affected by CT, although a dramatic reduction was observed with respect to the secreted proteins. Transferrin secreted into the culture medium was substantially non-glycosylated, judging by the absence of [3H]N-AcGlc. These studies suggested that the toxin preferentially affects the synthesis, and hence the secretion of glycoproteins, although it did not block the secretion of the proteins albumin and transferrin, as these did not accumulate intercellularly. Since transferrin labelled with [3H]leucine but not [3H]N-AcGlc is detected in the culture medium of hepatocytes exposed to CT, it was concluded that glycosylation of the protein is not required for secretion. This study shows that the effects of CT on protein synthesis and secretion in cultured hepatocytes are similar to those reported for tunicamycin (TM). 相似文献
3.
J. Glass M.T. Nunez S.H. Robinson 《Biochemical and biophysical research communications》1977,75(2):226-232
To ascertain whether transferrin need enter the reticulocyte to deliver its iron after the association of transferrin with the cell membrane, {125I, 59Fe-}labeled transferrin was covalently bound to Sepharose beads. Iron uptake from Sepharose-bound transferrin into rabbit reticulocytes was about 9% that from free transferrin while heme synthesis was more efficient at nearly 19%. Similar results were obtained with murine transferrin and murine reticulocytes.These results indicate that the entrance of transferrin inside the cell is not an obligatory step in the process of iron uptake in rabbit and murine reticulocytes. 相似文献
4.
A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule under the control of a mouse metallothionein (MT) promoter, was used as a model system to develop strategies to increase inducible recombinant protein production. Gene expression regulated by the MT promoter is induced by heavy metals (e.g. Zn+2 or Cd+2) in a dose dependent fashion. However, at high concentrations these metals are toxic to cells. Culture protocols which balance these counteractive effects are needed to maximize transferrin production. Fully induced cells produced up to 0.7 pg transferrin/cell·h, a 3-fold increase in production over uninduced levels. Cell growth was inhibited at Cd+2 dosages above 1 fmol/cell; prolinged exposure at this dosage was cytotoxic. Cell specific transferrin productivities decreased within 48 h following induction with Cd+2 although cell-associated Cd+2 levels remain high. Further addition of Cd+2 to cultures restored cell specific transferrin production rates. This suggests that cell associated Cd+2 is sequestered into a form which does not stimulate the MT promoter. Cd+2 dosing regimes which maintained cell associated Cd+2 concentrations between 0.2 and 0.35 fmol/cell ensured cell growth and high cell specific productivities which maximized final product titers. For routine batch culture, initial Cd+2 loadings of 0.8 fmol/cell gave near-maximum transferrin production levels. For extended culture, repeated small doses of 0.5 fmol/cell every 24 to 48 h maximized transferrin synthesis with this cell line. 相似文献
5.
Jonathan Glass Marco T. Nunez Siegmund Fischer Stephen H. Robinson 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,542(1):154-162
Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of 125I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 105 in non-induced cells and 9.28 ± 1.57 · 105 after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from 59Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of 59Fe or [2-14C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol 59Fe and 32 pmol [2-14C]glycine were incorporated into heme per 108 cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol 59Fe and 90 pmol [2-14C]glycine were incorporated into heme per 108 cells/h. (4) The rate of incorporation of 59Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol 59Fe/h per 108 cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis. 相似文献
6.
Significant differences in the glucocorticoid- and cyclic nucleotide-mediated regulation of the secretory glycoproteins, α-fetoprotein and transferrin, have been observed to develop in a mouse hepatoma cell line, Hepa-2, after many passages in culture. Treatment of low-passage cells with hydrocortisone (10?6m), N6,O2-dibutyryl cyclic AMP (10?3m), or 8-bromo-cyclic AMP (10?3m) results in 1.5-, 2- to 4-, and 5.5- to 6-fold increases, respectively, in the rates of synthesis and secretion of α-fetoprotein. As expected of secretory proteins, the ratio of synthesis to secretion is 1 and remains unaltered when treatment with hydrocoritsone, N6,O2-dibutyryl cyclic AMP, and 8-bromo-cyclic AMP causes a stimulation of synthesis and secretion. Similar studies showing that albumin and transferrin synthesis and secretion are also balanced in these low-passage cells have been published and indicate that the regulation of synthesis and secretion remains coupled in these low-passage cells. In high-passage Hepa-2 cells, however, we have shown that the relative rate of α-fetoprotein synthesis is higher than its rate of secretion and that the ratio of synthesis to secretion is 4. Similarly, the ratio of transferrin synthesis to secretion is 3.6, whereas it remains unaltered for albumin. When the high-passage cells are treated with N6,O2-dibutyryl cyclic AMP, there is a greater increase in the rate of secretion for both glycoproteins, resulting in a reduction of the ratio of synthesis to secretion from 4 to 1.63 for α-fetoprotein and from 3.6 to 2.3 for transferrin. This effect on the secretion of α-fetoprotein and transferrin is specific for the cyclic nucleotides and occurs only in high-passage cells. Hydrocortisone treatment causes an increase in α-fetoprotein synthesis and secretion. However, the ratio of synthesis to secretion increases from 3.96 in control to 5.5 in treated cells. Our studies show, therefore, that there is an increase in this ratio because of a slightly greater effect on synthesis which is not reflected in secretion. Similarly, hydrocortisone exerts a greater increase in transferrin synthesis than secretion and causes the ratio of synthesis to secretion to increase from 3.6 to 6.2. We propose that during continued subculturing a Hepa-2 variant is selected in which the regulation of serum glycoprotein synthesis and secretion is uncoupled. Furthermore, this effect is specific for secretory glycoproteins since the regulation of albumin synthesis and secretion by hydrocortisone and cyclic nucleotides remained unaltered. 相似文献
7.
Judith P. Bradshaw Julie Hatton David A. White 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,847(3)
Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [3H]leucine or [3H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [3H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [3H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [3H]mannose and [14C]leucine confirmed this. The decreased incorporation of [3H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does. 相似文献
8.
Zahur Zaman Marie-Jeanne Heynen Robrecht L. Verwilghen 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,632(4):553-561
Mechanism of transferrin iron uptake by rat reticulocytes was studied using 59Fe- and 125I-labelled rat transferrin. Whereas more than 80% of the reticulocyte-bound 59Fe was located in the cytoplasmic fraction, only 25–30% of 125I-labelled transferrin was found inside the cells. As shown by the presence of acetylcholine esterase, 10–15% of the cytoplasmic 125I-labelled transferrin might have been derived from the contamination of this fraction by the plasma membrane fragments. Electron microscopic autoradiography indicated 26% of the cell-bound 125I-labelled transferrin to be inside the reticulocytes. Both the electron microscopic and biochemical studies showed that the rat reticulocytes endocytosed their plasma membrane independently of transferrin. Sepharose-linked transferrin was found to be capable of delivering 59Fe to the reticulocytes. Our results suggest that penetration of the cell membrane by transferrin is not necessary for the delivery of iron and that, although it might make a contribution to the cellular iron uptake, internalization of transferrin reflects endocytotic activity of the reticulocyte cell membrane. 相似文献
9.
Edward S. Cole Jonathan Glass 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,762(1):102-110
Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·105molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·104±1.9·104 for cells in suspension and 6.6·104±2.3·104 for adherent cultured cells. The apparent association constants were 1.23·107 1·mol?1 and 3.4·106 1·mol?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe11 chelator) decreased the cell associated 59Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of 59Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis. 相似文献
10.
Three malignant hematopoietic cell lines were used in studies on cellular iron metabolism. Our results show that iron-carrying transferrin became bound to specific dimeric cell surface receptors. Iron accumulated within the cell with time, whereas intact transferrin was released back to the medium. Chloroquine and NH4Cl, known as pH-raising agents in vesicles of the lysosomal system, inhibited iron accumulation and transferrin binding in a dose-dependent manner. This suggests that the acid pH in endosomes leads to the cleavage of the iron-transferrin bonds. Transferrin degradation was not found, which leads us to suggest a process of ‘acid flushing’ for the dissociation of iron from transferrin without the involvement of endosome-lysosome fusion. Taken together, the data agree with the concept of receptor-mediated endocytosis, as described for many macromolecules. Iron was stored in ferritin in the cell types tested. Only a minor part (less than 15%) of the iron was bound in hemoglobin in the K-562 cell line. The relationship between iron stores and exogenously added iron in heme synthesis was investigated using a double labelling (55Fe/59Fe) technique. The results showed that exogenous iron was preferentially used before the iron stored in ferritin. The results are discussed in relation to various hypotheses on cellular iron uptake and transport. 相似文献
11.
The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change59Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of59Fe-125I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once59Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin. 相似文献
12.
Bifunctional chelating agents contain a metal chelating group and a second functional group that is usually chemically reactive in nature. They allow stable attachment of radioactive metal ions to proteins and other biological molecules, for use as radiopharmaceuticals and in other applications. Their chemical synthesis, attachment to proteins, and reaction with metals are briefly reviewed. Propertiesin vivo of chelate-tagged transferrin, chelate-tagged antibody to the transferrin cell-surface receptor, and111indium-transferrin are compared.This paper was presented at the symposium honoring Dr. Feeney's 70th birthday. 相似文献
13.
Michel Fischbach Hanwei Cao Miguel Diez Ibanez Christos Tsaconas Sami Alouani Frédéric Montandon Mohammed El Baraka Prudent Padieu Michel Dreano Martine Chessebeuf-Padieu 《Cell biology and toxicology》1991,7(4):327-345
Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-rasEJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 µg albumin and 0.32 µg transferrin per 106 cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminoflourene (2-AFAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHnneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 µg albumin and 11.0 µg transferrin per 106 cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGHper 106 cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 µg albumin and 11.7 µg transferrin per 106 cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AFAF per mg cell protien. Hence, Ha-ras
EJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.Abbreviations
BWE
basal Williams' medium E
- FBS
fetal bovine serum
-
F10 or F12
basal Ham's F10 or F12 medium
- Ha-ras
EJ
EJ allele of the human cellular ras oncogen of Harvey
-
hGH
human growth hormone
-
hsp
heat shock protein gene
-
LE-p
liposome encapsulated plasmid
- N-OH-2-AFAF
N-hydroxy-2-acetylaminofluorene
- RLECC
rat liver epithelial cell
- SF
serum-free
- SS
serum-supplemented
- UGG
serum substitute UGltroser G®
- 1-OH-, 3-OH-2-AFAFF
1-hydroxy-, 3-hydroxy-2-acetylaminofluorene
- 2-AFAF
2-acetylaminofluorene
- 2-AFF
2-aminofluorene 相似文献
14.
A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
J. Abra Watkins Marco-Tulio Nunez Victoria Gaete Osvaldo Alvarez Jonathan Glass 《The Journal of membrane biology》1991,119(2):141-149
Summary The kinetics of the separate processes of Fe2(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and59Fe,125I-labeled rabbit transferrin. The binding of59Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2×105
m
–1 sec–1. The rate of internalization of59Fe-transferrin was directly measured at 520±100 molecules of Fe2(III)-transferrin internalized/sec/cell with 250±43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe2(III)-transferrin internalization the flux of59Fe was followed through three compartments: internalized transferrin, membrane, and cytosol.A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17±2 sec and 34±5 sec, respectively. Apparent rate constants of 0.0075±0.002 sec–1 and 0.0343±0.0118 sec–1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112±0.0025 sec–1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe2(III)-transferrin that are distinct from the handling of transferrin. 相似文献
16.
Transferrin receptor expression by the human tumour cell lines CCRF-CEM leukaemia and PMC-22B melanoma was studied, measuring the specific binding of fluorescein isothiocyanate (FITC)-labelled transferrin using a fluorescence-activated cell sorter. By measuring the fluorescence of cells stained at subsaturating concentrations of conjugate it was possible to calculate the average numbers of receptors per cell and the binding affinity by Scatchard analysis. These values (1.9 × 105 binding sites/cell, KA 1.2 × 109 M?1 for CCRF-CEM during exponential growth and 6.9 × 104 binding sites/cell, KA 1.4 × 10?9 M?1 for PMC-22B) are in close agreement with previously published data obtained using radiolabelled transferrin. The present method, however, allowed the transferrin receptor expression of individual cells within a population to be measured and thus it has been possible to test the hypothesis that transferrin receptor is a marker for cycling cells. Frequency-distribution histograms of transferrin receptor showed a wide range of values for both cell lines during exponential growth. When the extreme ranges were sorted and the cells examined for cellular DNA content it was found that those with the highest transferrin receptor expression were enriched with cells in S, G2, and M phases of the cell cycle, whereas those with low transferrin receptor expression were mainly in G1. However, two-parameter-correlated dot plots of transferrin receptor expression versus DNA content showed there was considerable overlap between the ranges of receptor expression for the different cell cycle compartments. Using a stathmokinetic method we have measured the proportion of quiescent cells in fed plateau phase cultures. Transferrin receptor expression was downgraded under these growth conditions but, contrary to expectation, the decline affected the population uniformly, without the emergence of a distinct, transferrin receptor-negative subpopulation corresponding to the increasing proportion of quiescent cells. Thus, although transferrin receptor expression bears some relation to cell cycle phase and reflects the proliferative activity of populations of cells, it is incapable of identifying individual cells which are out of cycle. 相似文献
17.
Susanna M. Rybak Hennie R. Hoogenboom Dianne L. Newton Jef C. M. Raus Richard J. Youle 《Cell biochemistry and biophysics》1992,21(1-3):121-138
Members of the pancreatic ribonuclease (RNase) family have diverse activities toward RNA that could cause them to function
during host defense and physiological cell death pathways. This activity could be harnessed by coupling RNases to cell binding
ligands for the purpose of engineering them into cell-type specific cytotoxins. Therefore, the cytotoxic potential of RNase
was explored by linking bovine pancreatic ribonuclease A via a disulfide bond to human transferrin or antibodies to the transferrin
receptor. The RNase hybrid proteins were cytotoxic to K562 human erythroleukemia cells in vitro with an IC50 around 10−7
M, whereas>10−4
M of native RNase was required to inhibit protein synthesis. Cytotoxicity required both components of the conjugate since excess
transferrin or ribonuclease inhibitors added to the medium protected the cells from the transferrin-RNase toxicity. Importantly,
the RNase conjugates were found to have potent antitumor effects in vivo. Chimeric RNase fusion proteins were also developed.
F(ab′)2-like antibody-enzyme fusions were prepared by linking the gene for human RNase to a chimeric antitransferrin receptor heavy
chain gene. The antibody enzyme fusion gene was introduced into a transfectoma that secreted the chimeric light chain of the
same antibody, and cell lines were cloned that synthesized and secreted the antibody-enzyme fusion protein of the expected
size at a concentration of 1–5 ng/mL. Culture supernatants from clones secreting the fusion protein caused inhibition of growth
and protein synthesis toward K562 cells that express the human transferrin receptor but not toward a nonhuman derived cell
line. Since human ribonucleases coupled to antibodies also exhibited receptor mediated toxicities, a new approach to selective
cell killing is provided. This may allow the development of new therapeutics for cancer treatment that exhibit less systemic
toxicity and, importantly, less immunogenicity than the currently employed ligand-toxin conjugates. 相似文献
18.
The effect of concanavalin A on transferrin and iron uptake by reticulocytes was determined using rabbit reticulocytes and rabbit transferrin labelled with 59Fe and 125I and concanavalin A (ConA) labelled with 131I. In concentrations of 50–200 μg/ml ConA markedly inhibited iron uptake but did not inhibit transferrin uptake or release from the cells. ConA was itself taken up by rabbit blood cells in a manner similar to that of transferrin except that the uptake was not specific for reticulocytes but occurred also with mature erythrocytes. The inhibition of iron uptake by concanavalin and the uptake of concanavalin by the cells were both inhibited by α-methyl-d-mannoside. It is concluded that the effects observed were due to the binding of concanavalin to glycoproteins of the cell membrane, either by a direct interaction with transferrin receptors or by the production of a non-specific change in the structure of the membrane. 相似文献
19.
In this study, the incorporation of [(14)C]leucine into albumin and transferrin in early rat foetuses, vitelline plus amniotic membranes, chorioallantoic placenta and perinatal rat liver slices was measured and used to detect and compare the rates of synthesis of the two proteins. Albumin synthesis was detected in the body of foetuses from 13 days gestation onwards. Transferrin synthesis was detected only after day 15. Transferrin synthesis was demonstrable in the membranes but not in the chorioallantoic placenta of all the animals investigated, i.e. from 13 to 19 days gestation. Synthesis of albumin and transferrin by the liver of near-term and postnatal animals was shown to correlate with published data on the parenchymal cell number/unit wet wt. of liver. Near-term foetuses synthesized relatively more transferrin than albumin when compared with 10-day postnatal animals. The serum concentrations of the two plasma proteins were also determined. These increased before term whereas the rate of synthesis of albumin and transferrin declined. Postnatally, plasma albumin concentration increased but transferrin concentration decreased, yet the rates of synthesis of both proteins by the liver increased with age. This lack of correlation between the rates of synthesis of the two proteins and their respective plasma concentrations could be explained in part by their increased stability after birth. There was also evidence that the liver haemopoietic cells took up transferrin although they do not synthesize the protein. Thus the decrease in this population of cells during development could also contribute to the discrepancy between liver synthesis and serum concentrations of transferrin. 相似文献
20.
Premysl Ponka Herbert M. Schulman Ania Wilczynska 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,718(2):151-156
We have examined whether reticulocytes depleted of transferrin might incorporate 59Fe from 59Fe-labelled pyridoxan isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of 59Fe when incubated with 20–200 μM 59Fe-PIH. The amount of 59Fe incorporated with 200 μM 59Fe-PIH is equal to or higher than that taken up from transferrin at 20 μM 59Fe concentration. After 60 min about 60% of the 59Fe taken up by the cells is recovered in heme while the remainder is probably still bound to PIH. 1 mM succinyl acetone (a specific inhibitor of heme synthesis) inhibits PIH-mediated incorporation of 59Fe into heme by about 79% indicating that 59Fe from 59Fe-PIH is incorporated into de novo synthesized protoporphyrin. As is the case with transferrin, erythrocytes do not incorporate 59Fe from 59Fe-PIH. Pretreatment of reticulocytes with pronase does not inhibit their ability to incorporate 59Fe from 59Fe-PIH, suggesting that, unlike the uptake of Fe from transferrin, membrane receptors are not involved in the uptake of Fe-PIH by the cells. 相似文献