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Tannic acid (TA), a plant polyphenol, has been described as having antimutagenic, anticarcinogenic and antioxidant activities. Since it is a potent chelator of iron ions, we decided to examine if the antioxidant activity of TA is related to its ability to chelate iron ions. The degradation of 2-deoxyribose induced by 6 microM Fe(II) plus 100 microM H2O2 was inhibited by TA, with an I50 value of 13 microM. Tannic acid was over three orders of magnitude more efficient in protecting against 2-deoxyribose degradation than classical *OH scavengers. The antioxidant potency of TA was inversely proportional to Fe(II) concentration, demonstrating a competition between H2O2 and AT for reaction with Fe(II). On the other hand, the efficiency of TA was nearly unchanged with increasing concentrations of the *OH detector molecule, 2-deoxyribose. These results indicate that the antioxidant activity of TA is mainly due to iron chelation rather than *OH scavenging. TA also inhibited 2-deoxyribose degradation mediated by Fe(III)-EDTA (iron = 50 microM) plus ascorbate. The protective action of TA was significantly higher with 50 microM EDTA than with 500 microM EDTA, suggesting that TA removes Fe(III) from EDTA and forms a complex with iron that cannot induce *OH formation. We also provided evidence that TA forms a stable complex with Fe(II), since excess ferrozine (14 mM) recovered 95-96% of the Fe(II) from 10 microM TA even after a 30-min exposure to 100-500 microM H2O2. Addition of Fe(III) to samples containing TA caused the formation of Fe(II)n-TA, complexes, as determined by ferrozine assays, indicating that TA is also capable of reducing Fe(III) ions. We propose that when Fe(II) is complexed to TA, it is unable to participate in Fenton reactions and mediate *OH formation. The antimutagenic and anticarcinogenic activity of TA, described elsewhere, may be explained (at least in part) by its capacity to prevent Fenton reactions.

In proliferating non-erythroid cells, the expression of transferrin receptors (TfR) is negatively regulated by the amount of intracellular iron. Fe-dependent regulation of TfR occurs post-transcriptionally and is mediated by iron-responsive elements (IRE) located in the 3' untranslated region of the TfR mRNA. IREs are recognized by a specific cytoplasmic binding protein (IRE-BP) that, in the absence of Fe, binds with high affinity to TfR mRNA, preventing its degradation. While TfR numbers are positively correlated with proliferation in non-erythroid cells, in hemoglobin-synthesizing cells, their numbers increase during differentiation and are, therefore, negatively correlated with proliferation. This suggests a distinct regulation of erythroid TfR expression and evidence, as follows, for this was found in the present study. (a) With nuclear run-on assays, our experiments show increased TfR mRNA transcription following induction of erythroid differentiation of murine erythroleukemia (MEL) with Me2SO. (b) Me2SO treatment of MEL cells does not increase IRE-BP activity which is, however, increased in uninduced MEL cells by Fe chelators. (c) Following induction of MEL cells, there is an increase in the stability of TfR mRNA, whose level is only slightly affected by iron excess. (d) Heme-synthesis inhibitors, such as succinylacetone and isonicotinic acid hydrazide, which inhibit numerous aspects of erythroid differentiation, also inhibit TfR mRNA expression in induced MEL cells. However, heme-synthesis inhibition does not lead to a decrease in TfR mRNA levels in uninduced MEL cells. Thus, these studies indicate that TfR gene expression is regulated differently in hemoglobin synthesizing as compared to uninduced MEL cells.

Our previous research has demonstrated that in hemoglobin-synthesizing cells, as compared with nonerythroid cells, a step in iron transport from transferrin localized between the transferrin receptor and ferrochelatase is rate-limiting for the synthesis of heme. In this communication we report our more recent studies on the mechanisms involved in the regulation of the transferrin receptors and ferrochelatase in differentiating erythroid cells. Our studies indicate that transferrin receptor gene expression is regulated differently in hemoglobin synthesizing as compared with uninduced murine erythroleukemia (MEL) cells: 1) With nuclear run-on assays our experiments showed increased transferrin receptor mRNA transcription cells of MEL following induction of erythroid differentiation with dimethylsulfoxide (DMSO). 2) DMSO treatment of MEL cells does not increase iron-responsive element binding protein (IRE-BP) activity which is, however, increased in uninduced MEL cells by Fe chelators. 3) Following induction of MEL cells there is an increase in the stability of transferrin receptor mRNA whose level is only slightly affected by iron excess. Using murine ferrochelatase cDNA as a probe, two ferrochelatase transcripts having lengths of 2.9 kb and 2.2 kb were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in nonerythroid tissues and the 2.2 more predominant in spleen. In MEL cells, the 2.9 ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by DMSO there is a preferential increase in the 2.2 kb transcript which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Our further experiments indicate that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal and suggest that the preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression. These results provide further evidence for the idea that iron metabolism and heme synthesis are controlled by distinct mechanisms in erythroid versus nonerythroid cells.

The purpose of this study is to clarify the role of iron, transferrin, an iron-binding protein in vertebrate plasma, and transferrin receptors in cell proliferation. Transferrin, which is indispensable for most cells growing in tissue culture, is frequently referred to as a "growth factor". Proliferating cells express high numbers of transferrin receptors, and the binding of transferrin to their receptors that is needed for cells to initiate and maintain their DNA synthesis is sometimes regarded as analogous to other growth factor-receptor interactions. Although numerous previous experiments strongly indicate that the only function of transferrin in supporting cell proliferation is supplying cells with iron, they did not completely rule out some direct or signaling role transferrin receptors could play in cell proliferation. To address this issue, we exploited transferrin-receptor-deficient mutant Chinese hamster ovary (CHO) cells (McGraw, T. E., Greenfield, L., and Maxfield, F. R., 1987, J. Cell. Biol. 105, 207-214) in which various aspects of iron and transferrin metabolism in relation to their capacity to proliferate were investigated. Variant cells neither specifically bind transferrin nor do their extracts contain any detectable functional transferrin receptors, yet they proliferate and synthesize DNA with rates comparable to those observed with parent CHO cells. Desferrioxamine, an iron chelating agent, inhibits growth and DNA synthesis of both variant and control CHO cells. This inhibition can be fully alleviated, in both cell types, by ferric pyridoxal isonicotinoyl hydrazone, which can supply cells with a utilizable form of iron by a pathway not requiring transferrin and their receptors. Studies of 59Fe uptake and 125I-transferrin binding revealed that parent cells can take up iron by at least three mechanisms: from transferrin by receptor-dependent and -independent (nonspecific, nonsaturable, not requiring acidification) pathways and from inorganic iron salts (initially present in the medium as FeSO4). Although variant CHO cells are unable to acquire transferrin iron via the receptor pathway, two remaining mechanisms provide these cells with sufficient amounts of iron for DNA synthesis and cell proliferation. In conclusion, although transferrin receptors are dispensable in terms of their absolute requirement for proliferating cells, a supply of iron is still needed for their DNA synthesis. Transferrin-receptor-deficient CHO cells may be a useful model for investigating receptor-independent iron uptake from transferrin and nontransferrin iron sources.

Various types of proliferating cell are known to express transferrin receptors which are necessary for transferrin-mediated cellular iron uptake. Neither the mechanism nor the physiological role of transferrin receptor induction has been established with certainty; although it may reflect an increased cellular requirement for iron which is essential for ribonucleotide reductase, a key enzyme of DNA synthesis. The aim of this study was to examine murine mammary gland transferrin-receptor levels during gland development. As compared to virgin controls, total mammary gland transferrin receptors expressed on the basis of DNA, increase during pregnancy and lactation by 29- and 45-fold, respectively. However, on the basis of DNA, mammary gland ferritin, measured by radioimmunoassay, decreased by about 75% and 85% during pregnancy and lactation, respectively, indicating that the increased transferrin receptor levels probably do not lead to intracellular iron accumulation. When epithelial cells from mammary glands of pregnant mice were cultured in vitro transferrin receptor expression correlated with cell proliferation. These results suggest that normal mammary growth which occurs mainly in mammary epithelial cells is associated with a significant increase in transferrin receptor. Since transferrin receptor levels remain high during lactation they are not associated solely with tissue growth, but may also function in transporting iron during milk production.

The effect of three groups of metabolic inhibitors on the incorporation of Fe and release of bicarbonate from transferrin by rabbit reticulocytes was measured. Inhibitors which affect reticulocyte Fe and transferrin uptake to the same extent (sodium arsenite, N-ethylmaleimide and iodoacetamide); those which inhibit reticulocyte Fe uptake to a greater extent than transferrin uptake (NaN3, NaF, NaCN, rotenone, oligomycin, 2,4-dinitrophenol and cycloheximide); and compounds which after reticulocyte heme synthesis (CoCl2, isonicotinic acid hydrazide and hemin) were used. In each case the effect on Fe incorporation and bicarbonate release was the same Thus, additional evidence has been obtained for the idea that the reticulocyte-mediated release of Fe and bicarbonate from transferrin are tightly coupled. The results are consistent with the hypothesis that an enzymatic attack on transferrin-bound bicarbonate is involved in the removal of Fe from transferrin by erythroid cells.

Dimethylsulfoxide (DMSO) induces hemoglobin synthesis and erythroid differentiation of Friend erythroleukemia cells in vitro. Induction is accompanied by increased transferrin-binding activity which is necessary for the cellular acquisition of iron from transferrin for hemoglobin synthesis. There are Friend cell variants in which hemoglobin synthesis is not induced by DMSO unless exogenous hemin is also present. In this study we have compared the inducibility of transferrin receptors and iron incorporation in DMSO-inducible (745) and -uninducible (M-18 and TG-13) Friend cell lines. Cellular transferrin-binding sites were estimated by Scatchard analysis of data obtained from specific binding of [125I]transferrin by the cells. Our results show that unlike 745, DMSO treatment of the variant cell lines M-18 and TG-13 does not result in increased transferrin-binding activity. The number of transferrin-binding sites and the rate of iron uptake is similar in uninduced 745 and DMSO-treated M-18 and TG-13 cells. Although exposure of M-18 cells to DMSO and hemin induces hemoglobinization, this treatment does not cause induction of transferrin receptors. These results indicate that the primary defect in M-18 cells may be the uninducibility of transferrin receptors. We have also shown that exposure of 745 cells to hemin during DMSO treatment prevents the induction of transferrin receptors, suggesting that hemin may control the expression of transferrin receptors in erythroid cells.

The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2–14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2 –14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2–14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.

SUMMARY Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were measured in the cytosol extracted from developing effective soybean root nodules and compared to the activities found in cytosol extracted from ineffective soybean root nodules and soybean root tissue. In the cytosol of effective soybean root nodules glutamine synthetase activity was found to increase up to floral initiation while glutamate dehydrogenase activity decreased. Glutamate synthase activity was detected but it did not increase as much as glutamine synthetase. In ineffective nodule cytosol and root tissue much lower levels of glutamine synthetase and similar levels of glutamate dehydrogenase, as compared to effective nodule cytosol, were found but no glutamate synthase was detectable. The results indicated that glutamine synthetase/glutamate synthase cycle is operative for ammonia assimilation in effective soybean root nodules and glutamate dehydrogenase probably has no synthetic role.

Extract: Preterm newborns were found to be markedly hypotransferrinemie when compared with normal term infants. At birth the concentration of transferrin in sera from preterm infants of gestational age equal to or less than 32 weeks is 45% of that found in normal term infant sera. The preterm infant transferrin levels slowly rise so that 7–8 weeks after birth they are 78% of the level found in the sera of normal term infants. We also found that the serum transferrin concentrations at birth correlate with gestational age. Therefore, the transferrin levels postnatally in early preterm infants reflect postconceptional rather than postnatal age. Speculation: Since we found that early preterm newborns are markedly hypotransferrinemic, administration of iron to such infants during their first few postnatal months may be contraindicated. Harmful side effects of iron during this period might include an increased susceptibility to infection because of the known bacteriostatic and fungistatic activities of iron-free transferrin.

We have examined whether reticulocytes depleted of transferrin might incorporate 59Fe from 59Fe-labelled pyridoxal isonicotinoyl hydrazone (PIH). Transferrin-depleted reticulocytes showed a time-, temperature- and concentration-dependent incorporation of 59Fe when incubated with 20-200 microM 59Fe-PIH. The amount of 59Fe incorporated with 200 microM 59Fe-PIH is equal to or higher than that taken up from transferrin at 20 microM 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 70%, 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.

A model consisting of 59 Fe-labelled macrophages was developed for screening potential iron-chelating drugs. Mouse peritoneal macrophages, induced by previous intraperitoneal injections of 3% thioglycollate, were labelled in vitro by their exposure to immune complexes of 59 Fe-transferrin-antitransferrin antibody. Optimal conditions for macrophage labelling and subsequent 59 Fe release were established. Sixty-two aromatic hydrazones, the majority of which had iron binding structures similar to pyridoxal isonicotinoyl hydrazone, were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde, 2-hydroxy-1-naphthylaldehyde and 2-furaldehyde) with various acid hydrazides perpared by systematic substitutions on the benzene ring. These compounds were examined for their potential to stimulate 59 Fe release from 59 Fe-labelled macrophages and also from reticulocytes and hepatocytes loaded with non-heme 59 Fe. The majority of hydrazones derived from pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde seemed to be equally effective in both the macrophage and reticulocyte testing systems. However, the pyridoxal hydrazones were much more active in hepatocytes than the other groups of hydrzaones. Several compounds proved to be very potent in mobilizing 59 Fe. These included hydrazones derived from 2-hydroxyl-1-naphthylaldehyde and benzoic acid hydrazide, p -hydroxybenzoic acid hydrazide, 2-thiophenecarboxylic acid hydrazide, and also pyridoxal benzoyl hydrazone, pyridoxal m -fluorobenzyol hydrazone and pyridoxal 2-thiophenecarboxyl hydrazone.

Transferrin is essential for cell proliferation and it was suggested that it may trigger a proliferative response following its interaction with receptors, serving as a growth factor. However, since the only clearly defined function of transferrin is iron transport, it may merely serve as an iron donor. To further clarify this issue, we took advantage of an iron chelate, ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH), which we developed and previously demonstrated to efficiently supply iron to cells without using physiological transferrin receptor pathway. As expected, we observed that blocking monoclonal antibodies against transferrin receptors inhibited proliferation of both Raji and murine erythroleukemia cells. This inhibited cell growth was rescued upon the addition of Fe-SIH which was also shown to deliver iron to Raji cells in the presence of blocking anti-transferrin receptor antibodies. Moreover, blocking anti-transferrin receptor antibodies inhibited [3H]thymidine incorporation into DNA and this inhibition could be overcome by added Fe-SIH. In addition, Fe-SIH slightly stimulated, while SIH (an iron chelator) significantly inhibited, DNA synthesis in phytohemagglutinin-stimulated peripheral blood lymphocytes. Taken together, these results indicate that the only function of transferrin in supporting cell proliferation is to supply cells with iron.

LEGHAEMOGLOBIN (Lb), a myoglobin-like protein, is found in large quantities in the root nodules which result from the symbiotic association between the bacterium Rhizobium and plants of the family Leguminosae. The probable function of Lb is the regulation of intranodule oxygen tension1 and its presence is correlated with rhizobial nitrogenase2. The haem prosthetic group of Lb is of bacterial origin3 and the plant contains the genetic determinants for the apoprotein4,5. Soybean Lb consists of two major components of different electrophoretic mobility6. The faster component (LbF) is composed of 139 amino acids, has a molecular weight of 15,600 and contains an amino terminal glycine residue, while the slower component (LbS) is composed of 143 amino acids, has a molecular weight of 15,900 and contains an amino terminal valine residue. Because of these differences LbF and LbS are presumed to be different gene products7. Thus they are useful markers in studies on the transfer of plant genes involved in symbiosis to other plants.

In many types of cells the synthesis of delta-aminolevulinic acid (ALA) limits the rate of heme formation. However, results from our laboratory with reticulocytes suggest that the rate of iron uptake from transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis during erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe-SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels of Fe-Tf (20 microM). Furthermore, in induced Friend cells 100 microM Fe-SIH stimulated 2-14C-glycine incorporation into heme up to 3.6-fold as compared to the incorporation observed with saturating concentrations of Fe-Tf. In contrast, Fe-SIH, even when added in high concentrations, did not stimulate heme synthesis in uninduced Friend cells but was able to do so as early as 24 to 48 h following induction. In addition, contrary to previous results with rabbit reticulocytes, Fe-SIH also stimulated globin synthesis in induced Friend cells above the level seen with saturating concentrations of transferrin. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells.

Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.

14C-bicarbonate-labelled transferrin was prepared in order to study the role of bicarbonate in the cell-mediated release of iron from transferrin. 14C-bicarbonate bound to transferrin only in the presence of iron and with a ratio of bound bicarbonate to bound iron of one. The transferrin-14C-bicarbonate complex was very stable in Tris-HCl buffered at pH 7.5–9.0 even in the presence of excess non-radioactive bicarbonate. However, oxalate, citrate, and phosphate promoted a rapid exchange of transferrin-bound 14C-bicarbonate with bicarbonate present in the medium. Rabbit reticulocytes effected a temperature-dependent release of 14C-bicarbonate from transferrin at the same rate at which they incorporated 59Fe from transferrin — suggesting the existence of a coordinated mechanism in the cells for the release of both iron and bicarbonate from transferrin.

An in vitro system is described in which iron incorporation into rabbit reticulocytes and bone marrow cells from pure transferrin is qualitatively comparable to that found in vivo. The amount of stromal-bound iron reproducibly represents 2% and 12% of the total iron incorporated respectively into reticulocytes and bone marrow cells after 2.5 h of incubation. After 60 min of incubation only 40% of the reticulocyte stromal-bound iron behaved like a metabolic intermediate. All the metabolically active, membrane-bound iron could be accounted for a transferrin iron. Of the transferrin found in washed cells, 50–60% was recovered in the cytoplasmic fraciton, and was shown by isoelectric focusing to be undergraded. It is likely that it represents intracellular transferrin. The rate of hemoglobin synthesis was linear for at least 2.5 h in both reticulocytes and bone marrow cells, while bone marrow cells showed z greater iron incorporation into heme than reticulocytes. There was no detectabel low molecular weight iron in the cell supernatant of reticulocytes or bone marrow cells and hemoglobin-iron accounted for more than 92% of the total iron incorporated into the reticulocytes.