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Since there are several problems with desferrioxamine (DFO) therapy, pyridoxal isonicotinoyl hydrazone (PIH) has been studied for more than 10 years as a promising new candidate for iron chelation therapy in iron-overload diseases. Iron chelation could also be helpful for experimental treatment of several other pathologies including rheumatoid arthritis and heart ischemia/reperfusion, due to the generation of oxyradicals and lipid peroxidation mediated by delocalized iron. We demonstrate here that sub-millimolar levels of PIH can inhibit the Fe(III)-EDTA/ascorbate-mediated formation of hydroxyl-like radicals as tested by the release of ethylene from 2-keto-4-methylthiobutyric acid (KMB assay) and the formation of malonaldehyde from 2-deoxyribose damage. PIH could also decrease the rates of Fe(III)-EDTA-mediated oxidation of ascorbate and block the peroxidation of liposomes of rat brain phospholipids induced by ferrous iron-EDTA. In all cases the in vitro antioxidant effectiveness of PIH was comparable to its analogs-including salicylaldehyde isonicotinoyl hydrazone-and to DFO. We conclude that PIH and its analogs are effective new candidates against iron-mediated oxidative stress for use in experimental medicine.

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.

Pyridoxal isonicotinoyl hydrazone (PIH) has previously been studied for use in iron chelation therapy in iron-overload diseases. It is an efficient in vitro antioxidant due to its Fe(III) complexing activity (Schulman, H. M., et al. Redox Report 1:373-378; 1995). Pathologies associated with iron-overload include hepatic and other cancers. Since oxidative alterations of DNA can be linked to the development of cancer, we decided to study whether PIH protects DNA against in vitro oxidative stress. We report here that pUC-18 plasmid DNA is damaged by *OH radicals generated from Fe(II) plus H2O2 or from Fe(II) plus hypoxanthine/xanthine oxidase. The DNA damage was quantified by determining the diminution of supercoiled DNA forms after oxidative attack using agar gel electrophoresis. Micromolar amounts of PIH (20-30 microM) were able to half-protect DNA from iron (1-7.5 microM)-mediated *OH formation. The antioxidant capacity of PIH was significantly higher than that of some of its analogs and desferrioxamine. PIH and some of its analogues could also inhibit the oxidative degradation of 2-deoxyribose caused by Fenton reagents. Since we observed that PIH enhances the Fe(II) autoxidation rate, measured by the ferrozine technique, PIH may limit *OH formation and consequently DNA damage by decreasing the amount of Fe(II) available to catalyze 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.

Iron chelating agents are essential for treating iron overload in diseases such as beta-thalassemia and are potentially useful for therapy in non-iron overload conditions, including free radical mediated tissue injury. Deferoxamine (DFO), the only drug available for iron chelation therapy, has a number of disadvantages (e.g., lack of intestinal absorption and high cost). The tridentate chelator pyridoxal isonicotinoyl hydrazone (PIH) has high iron chelation efficacy in vitro and in vivo with high selectivity and affinity for iron. It is relatively non-toxic, economical to synthesize and orally effective. We previously demonstrated that submillimolar levels of PIH and some of its analogues inhibit lipid peroxidation, ascorbate oxidation, 2-deoxyribose degradation, plasmid DNA strand breaks and 5,5-dimethylpyrroline-N-oxide (DMPO) hydroxylation mediated by either Fe(II) plus H(2)O(2) or Fe(III)-EDTA plus ascorbate. To further characterize the mechanism of PIH action, we studied the effects of PIH and some of its analogues on the degradation of 2-deoxyribose induced by Fe(III)-EDTA plus ascorbate. Compared with hydroxyl radical scavengers (DMSO, salicylate and mannitol), PIH was about two orders of magnitude more active in protecting 2-deoxyribose from degradation, which was comparable with some of its analogues and DFO. Competition experiments using two different concentrations of 2-deoxyribose (15 vs. 1.5 mM) revealed that hydroxyl radical scavengers (at 20 or 60 mM) were significantly less effective in preventing degradation of 2-deoxyribose at 15 mM than 2-deoxyribose at 1.5 mM. In contrast, 400 microM PIH was equally effective in preventing degradation of both 15 mM and 1.5 mM 2-deoxyribose. At a fixed Fe(III) concentration, increasing the concentration of ligands (either EDTA or NTA) caused a significant reduction in the protective effect of PIH towards 2-deoxyribose degradation. We also observed that PIH and DFO prevent 2-deoxyribose degradation induced by hypoxanthine, xanthine oxidase and Fe(III)-EDTA. The efficacy of PIH or DFO was inversely related to the EDTA concentration. Taken together, these results indicate that PIH (and its analogues) works by a mechanism different than the hydroxyl radical scavengers. It is likely that PIH removes Fe(III) from the chelates (either Fe(III)-EDTA or Fe(III)-NTA) and forms a Fe(III)-PIH(2) complex that does not catalyze oxyradical formation.

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.

Iron ions play a central role in .OH radicals formation and induction of oxidative stress in living organisms. Iron-catalyzed .OH radical formation degrades deoxyribose to thiobarbituric acid reactive substances (TBA-RS). This paper analyzes kinetic properties of the Fe(III)-EDTA-catalyzed deoxyribose degradation in the presence of ascorbate. The yield of TBA-RS formation in the presence of EDTA was 4-fold higher than in its absence, contrasting with results reported elsewhere, Cu(II)-EDTA and Fe(III)-citrate were unable to catalyze deoxyribose degradation. The dependence on deoxyribose concentration was fitted to a Lineweaver Burk-like plot and it was calculated that approximately 4.5 mM deoxyribose scavenged half of the .OH radicals formed. The data for Fe(III)-EDTA concentration dependence could also be fitted to a rectangular hyperbolic function. This function was linear up to 1 microM added FeCl3 and this property could be utilized as an assay for the estimation of submicromolar iron concentrations. Submicromolar concentrations of iron could induce measurable yields of TBA-RS. Differences of as little as 0.1 microM Fe(III)-EDTA could be reproducibly detected under optimum experimental conditions, above a consistent background absorbance that was equivalent to 0.35 +/- 0.05 microM Fe(III)-EDTA and represented contaminating iron in the reactants that could not be removed with Chelex-100. The low method determination limit makes the deoxyribose degradation reaction potentially useful as a new, highly sensitive and cost effective assay for iron quantification.

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.

We have examined the changes in soybean leghemoglobins in relation to plant age, nodule size, and nitrogenase (EC 1.7.99.2) activity. The ratio of nitrogenase activity to leghemoglobin content declines throughout plant development and senescence. This results from a slower rate of increase of leghemoglobin compared with that of nitrogenase activity during early nodule development and a more rapid decline in nitrogenase activity after flowering. Larger nodules were generally found to have higher leghemoglobin contents but lower nitrogenase activity per unit fresh weight than nodules of intermediate size. The concentration of leghemoglobin in the nodules increases until the plants have flowered, at which time it represented 40% of the total soluble protein of the nodules. The proportions of the two major leghemoglobin species separable by electrophoresis (LbF and LbS) were measured and it was found that although LbF was the major component of all samples examined, the ratio of LbF:LbS decreased with increasing nodule size and age.

Poly(A)-containing leghemoglobin mRNA from soybean root nodules has been purified 84-fold, as judged by its ability to direct the in vitro synthesis of leghemoglobin in a wheat germ system. It has a poly(A) content of 8.6% and a molecular weight, estimated by formamide gel electrophoresis, of 260 000. mRNA with a molecular weight of around 143 000 would be sufficient to code for leghemoglobin. Thus, with respect to both its poly(A) content and its unexpectedly high molecular weight, leghemoglobin mRNA is similar to mRNAs isolated from animal tissues.

The chelating agent pyridoxal isonicotinoyl hydrazone (PIH) has recently been shown to mobilize 59Fe from reticulocytes loaded with non-heme 59Fe. In this study, various chelating agents were tested for their ability to effect the mobilization of iron from reticulocytes by PIH. They fall into several groups. The largest group includes chelators such as citrate, ethylenediaminetetracetic acid and desferrioxamine, which fail to affect PIH-induced iron mobilization and do not mobilize iron per se. Either these chelators do not enter reticulocytes or they do not take up iron from PIH-Fe complexes. The second group includes chelators such as 2,2'-bipyridine, 1,10-phenanthroline, bathophenanthroline sulfonate and N,N'-ethylenebis(o-hydroxyphenylglycine) which inhibit PIH-induced iron mobilization from reticulocytes and, when added together with PIH, induce radioiron accumulation in an alcohol-soluble fraction of reticulocytes. It appears that these chelators enter the cell and compete with PIH for 59Fe(II), but having bound iron are unable to cross the cell membrane. Spectral analysis suggests that Fe(II) chelators such as 2,2'-bipyridine and 1,10-phenanthroline remove iron from Fe(II)PIH but are not able to do so from Fe(III)PIH. Then there are compounds such as 2,3-dihydroxybenzoic acid and catechol which potentiate PIH-induced iron mobilization although they are unable to mobilize iron from reticulocytes by themselves. Lastly, there is a group of miscellaneous compounds which include chelators that either potentiate the iron-mobilizing effect of PIH as well as mobilizing iron from reticulocytes by themselves (tropolone), or that reduce PIH-induced iron mobilization while themselves having an iron-mobilizing effect (N,N'-bis(2,3-dihydroxybenzoyl)-1,6-diaminohexane). In further experiments, heme was found to stimulate globin synthesis in reticulocytes, the heme synthesis of which was inhibited by PIH, suggesting that PIH is probably not toxic to the 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.

Ferrihemoglobin can (1) stimulate globin synthesis in rabbit reticulocytes and in a reticulocyte cell-free protein synthesizing system, and (2) cause newly synthesized αβ-globin dimers to form tetramers which are indistinguishable from hemoglobin in the ultracentrifuge.These observations are consistent with the idea that a small pool of ferrihemoglobin may act as an intracellular heme carrier in reticulocytes.

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.

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-14C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 microMM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not delta-aminolevulinic acid, partly overcame the inhibition of 59Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits delta-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 microM 0.1 and 1 mM, inhibited 59Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte 59Fe uptake by about 25-30%. In succinylacetone-treated cells 59Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and 59Fe-transferrin-pretreated reticulocytes results in the transfer of 59Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.

Forty-eight strains of Rhizobium isolated from the root nodules of three species of legumes indigenous to the high tundra (Astragalus alpinus, Oxytropis maydelliana andOxytropis arctobia) are phenotypically heterogenous with respect to intrinsic antibiotic resistance, expression of nitrogenase activityex planta and plasmid content. All of the strains possess a 250–300 kb plasmid and are homologous to each other on the genomic DNA level but have little DNA homology with selected reference strains of well characterized species of rhizobia. The arctic rhizobia have an optimum growth temperature of 23°C and can grow slowly at 5°C. The DNA from four of the isolates, which were selected for detailed investigation, have sequences homologous tonif andnod genes fromRhizobium trifolii.

Forty-eight strains of rhizobia were isolated from the root nodules ofAstragalus alpinus (21),Oxytropis maydelliana (19) andOxytropis arctobia (8), three species of arctic legumes found in the Melville Peninsula, Northwest Territories, Canada. On the basis of 74 characteristics (cultural, physiological, biochemical and host nodulation range) the 48 arctic rhizobia could be divided into 11 distinct groups by numerical analysis techniques. All 48 arctic rhizobia were able to nodulate the three arctic legume species and also sainfoin (Onobrychis viciifolia), however, milkvetch (Astragalus cicer) was only nodulated by 33 strains. In general, the arctic rhizobia showed properties found in both Rhizobium and Bradyrhizobium. The adaptation of the arctic strains to low temperature is indicated by their ability to grow in liquid culture at 5°C.

Significant levels of nitrogenase activity (nitrogen fixation) were demonstrated in three species of Arctic legumes (Oxytropis maydelliana, O. arctobia and Astragalus alpinus) growing in high tundra at Sarcpa Lake, Melville Peninsula, N.W.T. Nitrogenase activity of intact plants was correlated with the number of nodules per plant, with field soil temperatures and limited by water shortage. Activity in freshly detached nodules showed a plateau of maximum activity between 10°C and 25°C and a near linear decline with temperature down to 0°C. Unusually, the segmented nodules of all three species are perennial in which growth and leghaemoglobin production resumes each spring from an overwintering apical meristem. Nodules are most numerous in the warmer soil stratum (2–10 cm. depth). Other studies indicate that the arctic rhizobia belong to a single cold-adapted species which has co-evolved with the legumes of tundra.

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.

To prevent autoxidation leghemoglobin was extracted from soybean root nodules of different ages in an atmosphere of carbon monoxide. Spectrophotometric examination of the leghemoglobins revealed that, during the functional life of the nodules, leghemoglobin exists only in its reduced form. Therefore, the decline of nitrogenase activity in older root nodules cannot be attributed to the accumulation of oxidized leghemoglobin.

The synthesis of heme and globin in rabbit reticulocytes was compared at 35 and 25 degrees C. The lower temperature decreased heme synthesis significantly more than globin synthesis and resulted in a much greater accumulation of globin dimers. After 16 h of incubation in the absence of iron, globin synthesis in reticulocytes which were at 35 degrees C could not be stimulated by iron, whereas cells which were at 25 degrees C responded with nearly control levels of globin synthesis. Since the formation of the hemin-controlled translational repressor in reticulocyte lysates is also decreased much more than protein synthesis at reduced temperature the results provide evidence for a physiological role for the translational repressor in controlling globin synthesis in reticulocytes.

1. 1. Heme synthesis from δ-amino[4- 14 C]levulinic acid in soybean root nodule extracts is inhibited by exogenous heme with maximum (50%) inhibition occurring at a heme concentration of 1 · 10 −4 M. Removal of the exogenous heme results in a reversal of this inhibition. 2. In the nodule extracts, heme synthesis from δ-amino[su14C]levulinic acid is stimulated by the addition of apo-leghemoglobin. We conclude that heme biosynthesis in soybean root nodules is endproduct, negatively feedback controlled, and we suggest that heme synthesis in this symbiotic complex may be coupled to that of apo-leghemoglobin.

Co2+ was employed as an inhibitor of heme synthesis in rabbit reticulocytes and bone marrow cells. Heme synthesis was measured using [2-14C]glycine and [4-14C]δ-aminolevulinate. Globin synthesis was measured using [2-14C]glycine, [14C]leucine and [14C]valine. Radioactive assays of partially purified globin revealed parallel rates of inhibition of heme and globin synthesis when the latter was labelled with [14C]leucine or [14C]valine. When [2-14C]glycine was used as the radioactive precursor of globin, globin synthesis was apparently stimulated by Co2+. However, when the amount of radioactivity in purified glycine labelled α-and β-globin chains was measured, it was found that globin synthesis was inhibited by Co2+. It was concluded that Co2+ does not dissociate heme and globin synthesis in erythroid cells when they are incubated in a medium optimal for hemoglobin synthesis, but rather inhibits both processes to the same extent. These results are relevant to the postulated regulatory role of heme in globin chain initiation.

By disc electrophoresis of leghemoglobins produced in the nodules of various legumes inoculated with specific strains of the bacterial genus Rhizobium, we have shown that: 1. 1. A single plant type treated with rhizobial strains of diverse origins always produces leghemoglobins having the same electrophoretic mobilities. 2. 2. Pairs of plant types produce effective nodules when inoculated with single rhizobial strains. The leghemoglobins are always plant-type specific. 3. 3. Representative species of the pea cross-inoculation group, effectively nod ulated with a single rhizobial strain, each produced a different electrophoretic pattern of leghemoglobin. These results are interpreted as evidence that the type of leghemoglobin produced in a given rhizobium-legume symbiosis, is plant specific. It is speculated that this effect is dependent on the genetic information, defining the amino acid sequence for apoleghemoglobins, being resident in the plant genome.

A defined medium for the study of rabbit reticulocyte maturation in vitro is described; the use of this medium led to the following observations. 1. 1. During maturation, hemoglobin synthesis itself had no effect on the rate of decay of the reticulocyte's ability to synthesize hemoglobin. 2. 2. Iron deprivation during maturation led to an irreversible loss in the reticulocyte's ability to utilize exogenous iron for hemoglobin synthesis. 3. 3. Addition of hemin to the maturation medium resulted in inhibition of globin synthesis in fresh cells, but stimulation in cells which had been incubated for 20 h. 4. 4. Comparison of whole cell and cell-free protein synthesizing rates of fresh cells and cells incubated for 20 h showed a much greater decline in activity of the whole cells than the cell-free systems derived from them. The conclusion drawn from these observations is that heme synthetic capacity declines more rapidly than globin synthetic capacity during maturation in vitro . This suggests that the duration of heme synthetic capacity may be a quantitatively controlling feature in the development of erythroid cells.

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.

LEGHAEMOGLOBIN (Lb), a haem protein with structural and functional similarities to animal myoglobin1, is found uniquely in legume root nodules,the sites of nitrogen fixation which form during symbiotic association between legumes and soil bacteria of the genus Rhizobium. Alhough in certain conditions free-living Rhizobium can express nitrogenase activity in the absence of Lb (see, for example, ref. 2), the presence of the protein seems to be required for the development of nitrogenase activity by Rhizobium bacteroids within legume root nodule cells. Lb probably functions by transporting oxygen to Rhizobium bacteroids, at the same time maintaining a low partial pressure of oxygen to protect nitrogenase from oxygen-inactivation.

Summary The oxidation state of newly synthesized hemoglobin in reticulocytes was determined by analyzing the hemoglobin from pulse-labelled cells by isoelectric focusing in acrylamide gels. The newly synthesized material contained oxidized heme groups when either 14C-leucine or 59Fe was used for the radioactive pulse. With both isotopes the radioactive hemoglobin rapidly equilibrated with intracellular ferrohemoglobin during subsequent incubation of the cells in non-radioactive medium. The results suggest that ferrihemoglobin is an intermediate in the biosynthesis of functional ferrohemoglobin in reticulocytes.

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.

Cobalt was found to stimulate globin synthesis in reticulocytes the heme synthesis of which was inhibited by isonicotinic acid hydrazide or 4,6-dioxoheptanoic acid (succinyl acetone). These results suggest that in reticulocytes cobalt does not stimulate globin synthesis through the formation of cobalt protoporphyrin. They do, how-ver, support the hypothesis that cobalt causes the formation of a pool of free heme from preformed hemoglobin which stimulates the synthesis of new globin chains.

Iron, which is involved in many metabolic processes including transport and storage of oxygen and oxidation-reduction reactions is an essential element for all living cells with the one possible exception lactic acid bacilli. However, organisms are not equipped with active excretory systems for iron and, therefore, if excessive amounts of iron get to the organism they accumulate without being excreted. This results in an iron overload which is a common finding in patients with refractory anemias such as thalassemia major, some other hemoglobinopathies and certain other anemias1 – 4. The overload is a consequence of increased iron absorption, but primarily of long-term transfusion therapy. Excess iron accumulates in liver, heart and endocrine glands causing serious dysfunction and death. Excess iron is removed from such patients by administration of chelating agents, primarily desferrioxamine. However, since desferrioxamine is inadequate and expensive, new iron chelating agents are needed5.

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.

To elucidate a mechanism for cobalt-stimulated globin synthesis in rabbit reticulocytes, the cells were incubated with 1 mM CoCl2 containing 60Co. Although the cells incorporated cobalt irreversibly, evidence for the synthesis of cobalt-protoporphyrin was not obtained. More than 90% of the cobalt recovered in the cell cytoplasma was bound to the globin moiety of hemoglobin. Similar results were obtained with erythrocytes and purified hemoglobin incubated with 60CoCl2. By CM-Sephadex chromatography and isoelectric focusing it was found that cobalt-labelled hemoglobin differs slightly in charge from native hemoglobin, possessing a lower isoelectric point. It was also demonstrated that the heme in cobalt-labelled hemoglobin is less firmly bound to globin than the heme in the native protein, and that the purified cobalt-treated protein can stimulate globin synthesis in rabbit reticulocytes. It is postulated that in reticulocytes cobalt stimulates globin synthesis indirectly by labilizing intracellular hemoglobin.

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.

The soluble, labeled product of a cell-free system derived from rabbit reticulocytes was investigated to determine its role in the assembly of hemoglobin (Hb). It has been concluded that the cell-free system synthesizes a protein which by two independent criteria is not yet a finished Hb molecule. The product was found to be more basic than Hb on ion-exchange chromatography and to have a molecular weight, as determined by gel filtration and sucrose density centrifugation, characteristic of a dimer. The dimer is unstable but can be converted, in part, to a stable Hb-like molecule by exogenous hemin. These findings suggest that the dimer is an intermediate in the synthesis of Hb. The significance of this is discussed in relation to the regulation of Hb synthesis.

A method is described for the preparation of concentrated suspensions of viable rabbit reticulocytes from animals rendered anemic by repeated bleeding. The method involves the centrifugation of washed red blood cells through dextran density gradients.

By separating breis derived from soybean plant root nodules into soluble and particulate components and incubating each with δ-amino[4−14C]levulinic acid, it was found tha: (I) The heme synthesized by the nodules is principally the product of the particulate component. (2) The particulate component is predominantly bacteriods and is devoid of plant mitochondria. (3) Some of the heme synthesized in the breis is incorporated into leghemoglobin. We conclude that bacteroids in the nodules are responsible for the major part of the synthesis of heme for leghemoglobin.