An evolutionarily ancient mechanism for regulation of hemoglobin expression in vertebrate red cells.

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution.

Low hemoglobin levels are independently associated with neonatal acute kidney injury: a report from the AWAKEN Study Group.

BACKGROUND: Studies in adults showed a relationship between low hemoglobin (Hb) and acute kidney injury (AKI). We performed this study to evaluate this association in newborns. METHODS: We evaluated 1891 newborns from the Assessment of Worldwide AKI Epidemiology in Neonates (AWAKEN) database. We evaluated the associations for the entire cohort and 3 gestational age (GA) groups: <29, 29-<36, and ≥36 weeks' GA. RESULTS: Minimum Hb in the first postnatal week was significantly lower in neonates with AKI after the first postnatal week (late AKI). After controlling for multiple potential confounders, compared to neonates with a minimum Hb ≥17.0 g/dL, both those with minimum Hb ≤12.6 and 12.7-14.8 g/dL had an adjusted increased odds of late AKI (aOR 3.16, 95% CI 1.44-6.96, p = 0.04) and (aOR 2.03, 95% CI 1.05-3.93; p = 0.04), respectively. This association was no longer evident after controlling for fluid balance. The ability of minimum Hb to predict late AKI was moderate (c-statistic 0.68, 95% CI 0.64-0.72) with a sensitivity of 65.9%, a specificity of 69.7%, and a PPV of 20.8%. CONCLUSIONS: Lower Hb in the first postnatal week was associated with late AKI, though the association no longer remained after fluid balance was included. IMPACT: The current study suggests a possible novel association between low serum hemoglobin (Hb) and neonatal acute kidney injury (AKI). The study shows that low serum Hb levels in the first postnatal week are associated with increased risk of AKI after the first postnatal week. This study is the first to show this relationship in neonates. Because this study is retrospective, our observations cannot be considered proof of a causative role but do raise important questions and deserve further investigation. Whether the correction of low Hb levels might confer short- and/or long-term renal benefits in neonates was beyond the scope of this study.

Use of face mask by blood donors during the COVID-19 pandemic: Impact on donor hemoglobin concentration: A bane or a boon.

BACKGROUND: COVID-19 virus has caused the world's deadliest pandemic. Early April 2020, the Delhi Government made it compulsory for people to wear face masks while going outdoors to curb disease spread. Prolonged use of surgical masks during the pandemic has been reported to cause many adverse effects. Intermittent hypoxia has been shown to activate erythropoietin (EPO leading to increased hemoglobin mass. AIM: To analyze whether face mask induced intermittent hypoxia has any effect on the hemoglobin levels of healthy blood donors. MATERIALS AND METHODS: We retrospectively analyzed donor data from 1st July 2019-31st December 2020 for hemoglobin distribution across hemoglobin ranges and donor deferral on basis of hemoglobin. Study population was divided into two cohorts Group 1- (1st July 2019-31 st March 2020): before implementation of mandatory face masks Group 2- (1st April 2020-31 st December 2020): after implementation of mandatory face masks RESULTS: Mean Hb of blood donors in Group 2 (15.01 ± 1.1 g/dl) was higher than Group1 (14.49 ± 1.15 g/dl), (p < 0.0001). 47.1 % group2 donors had Hb of 16.1-18 g/dl compared to group1 (38.4 %). 52.9 % group 2 donors had Hb between 12.5-15 g/dl compared to 61.6 % Group 1 (p < 0.05). Deferral due to anemia was lesser in group 2 compared to group 1 (p < 0.00001). Group 2 had significantly higher deferral due to high Hb (>18 gm/dl) was than Group 1 (p = 0.0039). CONCLUSION: This study including 19504 blood donors spanning over one and a half year shows that prolonged use of face mask by blood donors may lead to intermittent hypoxia and consequent increase in hemoglobin mass.

Effect of lead exposure from electronic waste on haemoglobin synthesis in children.

BACKGROUND: Primitive electronic waste (e-waste) recycling is ongoing in Guiyu, so toxic heavy metals may continue to threaten the health of children in the area. OBJECTIVE: This study primarily aimed to evaluate the effect of e-waste exposure on haemoglobin (Hb) synthesis in preschool children. METHODS: Medical examinations were conducted with the permission of children's guardians and the approval of the Ethics Committee of the Medical College of Shantou University. This study recruited 224 children (aged 3-6 years, exposed group) who lived in Guiyu and 204 children (aged 3-6 years, control group) who lived in a town free of e-waste pollution. Blood levels of lead, Hb, ferritin, folate and vitamin B12 were tested in all children. Furthermore, all children were assessed for thalassemia, and their parents were asked to fill in questionnaires. RESULTS: There were no significant differences in the level of ferritin, folate, or vitamin B12 between the exposed and control groups (P > 0.05). No children were identified as having thalassemia in all study participants. Blood lead level (BLL) and the risk of children with BLL ≥ 10 µg/dL in the exposed group were significantly higher than those in the control group (all P < 0.01). Three subgroups of each group were created according to BLL (Group A: < 5.0 µg/dL; Group B: 5.0-9.9 µg/dL; Group C: ≥ 10.0 µg/dL). Hb level decreased with elevated BLL in the exposed group (P = 0.03), but not in the control group (P = 0.14). Hb levels in group B and group C were also significantly lower in the exposed group than in the control group (Group B: 122.6 ± 9.5 g/L versus 125.8 ± 8.2 g/L, P = 0.01; Group C: 120.3 ± 7.3 g/L versus 123.6 ± 8.3 g/L, P = 0.03). In addition, the prevalence of anaemia associated with BLLs above 10 µg/dL and between 5.0 and 9.9 µg/dL were both significantly higher in the exposed group than in the control group (4.0% vs. 0.5%, 5.4% vs. 1.5%, respectively, both P < 0.05). CONCLUSION: Lead exposure more significantly inhibits Hb synthesis in children who live in e-waste dismantling areas than in those who live in non-e-waste dismantling areas. Other toxins released from e-waste may also contribute to the inhibition of Hb synthesis and may lead to anaemia in local children. Further investigations are needed to provide evidence for the development of relevant protective measures.

Hsp90 chaperones hemoglobin maturation in erythroid and nonerythroid cells.

Maturation of adult (α2ß2) and fetal hemoglobin (α2γ2) tetramers requires that heme be incorporated into each globin. While hemoglobin alpha (Hb-α) relies on a specific erythroid chaperone (alpha Hb-stabilizing protein, AHSP), the other chaperones that may help mature the partner globins (Hb-γ or Hb-ß) in erythroid cells, or may enable nonerythroid cells to express mature Hb, are unknown. We investigated a role for heat-shock protein 90 (hsp90) in Hb maturation in erythroid precursor cells that naturally express Hb-α with either Hb-γ (K562 and HiDEP-1 cells) or Hb-ß (HUDEP-2) and in nonerythroid cell lines that either endogenously express Hb-αß (RAW and A549) or that we transfected to express the globins. We found the following: (i) AHSP and hsp90 associate with distinct globin partners in their immature heme-free states (AHSP with apo-Hbα, and hsp90 with apo-Hbß or Hb-γ) and that hsp90 does not associate with mature Hb. (ii) Hsp90 stabilizes the apo-globins and helps to drive their heme insertion reactions, as judged by pharmacologic hsp90 inhibition or by coexpression of an ATP-ase defective hsp90. (iii) In nonerythroid cells, heme insertion into all globins became hsp90-dependent, which may explain how mixed Hb tetramers can mature in cells that do not express AHSP. Together, our findings uncover a process in which hsp90 first binds to immature, heme-free Hb-γ or Hb-ß, drives their heme insertion process, and then dissociates to allow their heterotetramer formation with Hb-α. Thus, in driving heme insertion, hsp90 works in concert with AHSP to generate functional Hb tetramers during erythropoiesis.

Alpha and beta-Thalassemia mutations in Hubei area of China.

BACKGROUND: Thalassemia is a group of inherited hemoglobic disorders resulting from defects in the synthesis of one or more of the hemoglobin chains, which is one of the most prevalent inherited disorders in southern China. Only few studies reported the molecular characterization of α- and ß-Thalassemia in Hubei Province in the central of China. METHODS: A total of 4889 clinically suspected cases of thalassemia were analyzed by Gap-PCR, PCR-based reverse dot blot (RDB). RESULTS: 1706 (33.8%) subjects harbored thalassemia mutations, including 539 (11.0%) subjects with α-thalassemia, 1140 (23.3%) subjects with ß-thalassemia mutations, and 25 (0.51%) subjects with both α- and ß-thalassemia mutations. Seven genotypes of α-thalassemia mutations and 29 genotypes of ß-thalassemia mutations were characterized. --SEA/αα (66.05%), -α3.7/αα (24.12%), and -α4.2/αα (3.71%) accounted for 93.88% of the α-thalassemia mutations. ßIVS-II-654/ßN, ßCD41-42/ßN, ßCD17/ßN, ßCD27-28/ßN, ßCD71-72/ßN, ß - 28/ßN, ß - 29/ßN, ßCD43/ßN, ßE/ßN, accounting for 96.40% of all ß-thalassemia genotypes. Furthermore, mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) were sensitive markers for both ß-thalassemia and α-thalassemia with --SEA/αα, but not -α3.7/αα and -α4.2/αα. CONCLUSIONS: Our data indicated great heterogeneity and extensive spectrum of thalassemias in Hubei province of China.

Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis.

During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.

Enhancing hemoglobin peptide production from chicken blood fermentation by food-grade nonionic surfactant.

This study is focused on employing a potential process technology for enhancing hemoglobin peptides production from chicken blood. Effects of surfactants on chicken blood biodegradation and hemoglobin polypeptide accumulation were evaluated and the bioconversion conditions were optimized. Results suggested that surfactants exhibited the positive effect on hemoglobin peptides production during chicken blood bioconversion by Aspergillus niger. Dodecyl glucopyranoside was selected as the optimal surfactant and added at the 48th hour of the fermentation process (64 H) at the concentration of 6.0 g/L. Under the optimized conditions, 104.5 mg·N/mL amino nitrogen, 638.3 mg·N/mL nonprotein nitrogen, and 766.3 mg·N/mL soluble nitrogen were detected, which increased by approximately 0.7-, 3.7-, and 3.8-fold, respectively, compared with the control. Furthermore, the acid protease stability was remarkably intensified and the accumulated peptides were mainly distributed at 500-2,000 Da. Results from this work corroborate the potential of applying dodecyl glucopyranoside in hemoglobin polypeptide production from chicken blood.

National Iron Plus Initiative: Current status & future strategy.

Anaemia is a severe public health problem amongst all vulnerable age groups in India. The National Nutritional Anaemia Prophylaxis Programme initiated in 1970, was revised and expanded to include beneficiaries from all age groups namely children aged 6-59 months, 5-10 yr, adolescents aged 10-19 yr, pregnant and lactating women and women in reproductive age group under the National Iron Plus Initiative (NIPI) programme in 2011. The dose of iron, frequency and duration of iron supplementation and roles and responsibilities of the functionaries were described. At present, the coverage of beneficiaries with iron and folic acid has been poor at the national level. The prevalence of anaemia has continued to remain high during the last 60 years, and there has been no significant change in the scenario due to various reasons. The constraints in implementation and measures to improve the NIPI programme are discussed in the current article.

MS-275 Chemical Analogues Promote Hemoglobin Production and Erythroid Differentiation of K562 Cells.

ß-Thalassemia (ß-thal) is a hemoglobinopathy characterized by reduced or absent ß-globin production. Pharmacological reactivation of the γ-globin gene for the production of fetal hemoglobin (Hb F) presents an attractive treatment strategy. In an effort to identify promising therapeutic agents, we evaluated 80 analogues of the histone deacetylase inhibitor MS-275, a known Hb F inducer. The chemical analogues were identified via molecular modeling and targeted chemical modifications. Nine novel agents exhibited significant hemoglobin (Hb)-inducing and erythroid differentiation activities in the human K562 erythroleukemia cell line. Five of them appeared to be stronger inducers than the lead compound, MS-275, demonstrating the effectiveness of our method.

The Chaperones Involved in Hemoglobin Synthesis Take the Spotlight: Analysis of AHSP in the Argentinean Population and Review of the Literature.

Hemoglobin (Hb) synthesis is a complex, well-coordinated process that requires molecular chaperones. These intervene in different steps: regulating epigenetic mechanisms necessary for the adequate expression of the α- and ß-globin clusters, binding the nascent peptides and helping them acquire their native structure, preventing oxidative damage by free globin chains and preventing the cleavage of essential erythroid transcription factors. This study analyzed the distribution of the single nucleotide polymorphism (SNP) rs4296276 in intron 1 of the α-globin chaperone α Hb-stabilizing protein (AHSP) in the Argentinean population. The risk allele was found in thalassemia patients who exhibited more severe phenotypes than expected. Future studies may help establish the role of these chaperones as modifiers in pathological states with globin chain imbalance, such as thalassemia.

Extracellular glycine is necessary for optimal hemoglobinization of erythroid cells.

Vertebrate heme synthesis requires three substrates: succinyl-CoA, which regenerates in the tricarboxylic acid cycle, iron and glycine. For each heme molecule synthesized, one atom of iron and eight molecules of glycine are needed. Inadequate delivery of iron to immature erythroid cells leads to a decreased production of heme, but virtually nothing is known about the consequence of an insufficient supply of extracellular glycine on the process of hemoglobinization. To address this issue, we exploited mice in which the gene encoding glycine transporter 1 (GlyT1) was disrupted. Primary erythroid cells isolated from fetal livers of GlyT1 knockout (GlyT1-/-) and GlyT1-haplodeficient (GlyT1+/-) embryos had decreased cellular uptake of [2-14C]glycine and heme synthesis as revealed by a considerable decrease in [2-14C]glycine and 59Fe incorporation into heme. Since GlyT1-/- mice die during the first postnatal day, we analyzed blood parameters of newborn pups and found that GlyT1-/- animals develop hypochromic microcytic anemia. Our finding that Glyt1-deficiency causes decreased heme synthesis in erythroblasts is unexpected, since glycine is a non-essential amino acid. It also suggests that GlyT1 represents a limiting step in heme and, consequently, hemoglobin production.

Highly efficient production of hyaluronic acid by Streptococcus zooepidemicus R42 derived from heterologous expression of bacterial haemoglobin and mutant selection.

UNLABELLED: During Streptococcus zooepidemicus fermentation, most carbon sources are used to synthesize lactic acid, which can inhibit strain growth and hyaluronic acid production. Here, we expressed bacterial haemoglobin (Vhb) in Strep. zooepidemicus. Due to highly efficient oxygen use, only 15·26 g l(-1) lactic acid was produced, which is 0·73 times the quantity produced by the control strain. Compared with the control strain (1·61 g l(-1) ), hyaluronic acid (HA) production in this strain did not substantially increase, only to 2·16 g l(-1) . Next, we used a series of N-methyl-N'-nitro-N-nitroso-guanidine (NTG) treatments and selection programmes. Finally, we generated a hyaluronidase-negative and rifampin-resistant mutant strain that produces high levels of HA. The optimum carbon concentration for maximum hyaluronic acid production is only 30 g l(-1) of sucrose, which is lower than the control strain (60 g l(-1) ). The oxygen transfer rate coefficient KL a increased significantly to 372 ± 53 h(-1) from 18 ± 4 h(-1) of the control. The optimum carbon source for this strain is 21 g l(-1) of sucrose, 9 g l(-1) of maltose and 5 g l(-1) of glutamic acid. Hyaluronic acid accumulated at 6·7 g l(-1) in the culture broth. However, the molecular weight of HA decreased from 1835 KDa (Control) to 429 kDa. The prepared low-molecular weight HA could function as potential antiangiogenic substances, antiviral and antitumour agents to possibly be used as functional food ingredients. SIGNIFICANCE AND IMPACT OF THE STUDY: Hyaluronic acid (HA) has been used for a wide range of applications in health, cosmetic and clinical fields. During fermentation of Streptococcus to produce HA, 80-85% of the carbon source is used to produce lactic acid and acetic acid, and only approx. 5 and 10% of the carbon source is used to produce HA and biomass respectively. Here, we expressed bacteria haemoglobin (Vhb) in Streptococcus zooepidemicus, which can dramatically inhibit lactic acid production. After NTG treatments and selection programmes, we identified a mutant strain with highly efficient hyaluronic acid production (6·7 g l(-1) ) under economic fermentation conditions.

Aberrant splicing of genes involved in haemoglobin synthesis and impaired terminal erythroid maturation in SF3B1 mutated refractory anaemia with ring sideroblasts.

Refractory anaemia with ring sideroblasts (RARS) is distinguished by hyperplastic inefficient erythropoiesis, aberrant mitochondrial ferritin accumulation and anaemia. Heterozygous mutations in the spliceosome gene SF3B1 are found in a majority of RARS cases. To explore the link between SF3B1 mutations and anaemia, we studied mutated RARS CD34(+) marrow cells with regard to transcriptome sequencing, splice patterns and mutational allele burden during erythroid differentiation. Transcriptome profiling during early erythroid differentiation revealed a marked up-regulation of genes involved in haemoglobin synthesis and in the oxidative phosphorylation process, and down-regulation of mitochondrial ABC transporters compared to normal bone marrow. Moreover, mis-splicing of genes involved in transcription regulation, particularly haemoglobin synthesis, was confirmed, indicating a compromised haemoglobinization during RARS erythropoiesis. In order to define the phase during which erythroid maturation of SF3B1 mutated cells is most affected, we assessed allele burden during erythroid differentiation in vitro and in vivo and found that SF3B1 mutated erythroblasts showed stable expansion until late erythroblast stage but that terminal maturation to reticulocytes was significantly reduced. In conclusion, SF3B1 mutated RARS progenitors display impaired splicing with potential downstream consequences for genes of key importance for haemoglobin synthesis and terminal erythroid differentiation.

Anti-hepcidin therapy for iron-restricted anemias.

In this issue of Blood, Cooke et al demonstrate the potential of a fully human anti-hepcidin antibody as a novel therapeutic for iron-restricted anemias such as anemia of inflammation, cancer, or chronic kidney disease (formerly known as "anemia of chronic diseases").

A fully human anti-hepcidin antibody modulates iron metabolism in both mice and nonhuman primates.

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

Prevalence and determinants of declining versus stable hemoglobin levels in whole blood donors.

BACKGROUND: A too short recovery time after blood donation results in a gradual depletion of iron stores and a subsequent decline in hemoglobin (Hb) levels over time. This decline in Hb levels may depend on individual, unobserved characteristics of the donor. STUDY DESIGN AND METHODS: We used a data set of 5388 Dutch blood donors from the Donor InSight study. The statistical analysis is based on a Bayesian growth mixture model, which assumes that each donor belongs to one of several groups. Each group implies a different Hb trajectory, and donors with similar longitudinal trajectories belong to the same group. Analyses were performed for male and female donors separately. RESULTS: For both sexes the model identified four groups of donors. Stable Hb trajectories were found among 14% of male donors and 15% of female donors; declining Hb trajectories were observed in the remaining groups of donors. The percentage of donor deferrals differed strongly between groups. CONCLUSION: The model can be used to predict to which group a donor belongs, and this prediction can be updated after each donation. This is of high practical importance because early identification of donors with declining Hb levels could help to tailor donation intervals and to prevent iron deficiency and donor deferrals.

Higher iron pearl millet (Pennisetum glaucum L.) provides more absorbable iron that is limited by increased polyphenolic content.

BACKGROUND: Our objective was to compare the capacity of iron (Fe) biofortified and standard pearl millet (Pennisetum glaucum L.) to deliver Fe for hemoglobin (Hb)-synthesis. Pearl millet (PM) is common in West-Africa and India, and is well adapted to growing areas characterized by drought, low-soil fertility, and high-temperature. Because of its tolerance to difficult growing conditions, it can be grown in areas where other cereal crops, such as maize, would not survive. It accounts for approximately 50% of the total world-production of millet. Given the widespread use of PM in areas of the world affected by Fe-deficiency, it is important to establish whether biofortified-PM can improve Fe-nutriture. METHODS: Two isolines of PM, a low-Fe-control ("DG-9444", Low-Fe) and biofortified ("ICTP-8203 Fe",High-Fe) in Fe (26 µg and 85 µg-Fe/g, respectively) were used. PM-based diets were formulated to meet the nutrient requirements for the broiler (Gallus-gallus) except for Fe (Fe concentrations were 22.1±0.52 and 78.6±0.51 µg-Fe/g for the Low-Fe and High-Fe diets, respectively). For 6-weeks, Hb, feed-consumption and body-weight were measured (n = 12). RESULTS: Improved Fe-status was observed in the High-Fe group, as suggested by total-Hb-Fe values (15.5±0.8 and 26.7±1.4 mg, Low-Fe and High-Fe respectively, P<0.05). DMT-1, DcytB, and ferroportin mRNA-expression was higher (P<0.05) and liver-ferritin was lower (P>0.05) in the Low-Fe group versus High-Fe group. In-vitro comparisons indicated that the High-Fe PM should provide more absorbable-Fe; however, the cell-ferritin values of the in-vitro bioassay were very low. Such low in-vitro values, and as previously demonstrated, indicate the presence of high-levels of polyphenolic-compounds or/and phytic-acid that inhibit Fe-absorption. LC/MS-analysis yielded 15 unique parent aglycone polyphenolic-compounds elevated in the High-Fe line, corresponding to m/z = 431.09. CONCLUSIONS: The High-Fe diet appeared to deliver more absorbable-Fe as evidenced by the increased Hb and Hb-Fe status. Results suggest that some PM varieties with higher Fe contents also contain elevated polyphenolic concentrations, which inhibit Fe-bioavailability. Our observations are important as these polyphenols-compounds represent potential targets which can perhaps be manipulated during the breeding process to yield improved dietary Fe-bioavailability. Therefore, the polyphenolic and phytate profiles of PM must be carefully evaluated in order to further improve the nutritional benefit of this crop.

Characterization of THB1, a Chlamydomonas reinhardtii truncated hemoglobin: linkage to nitrogen metabolism and identification of lysine as the distal heme ligand.

The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO(•)) in either the ferric or ferrous state and has efficient NO(•) dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway.