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Author(s): Jeffrey B. McQuaid [1, 2]; Adam B. Kustka [3]; Miroslav Obornk [4, 5]; Ale Hork [4, 5]; John P. McCrow [1]; Bogumil J. Karas [1]; Hong Zheng [1]; Theodor [...]

Background/Objectives: To assess the iron status among European adolescents through selected biochemical parameters in a cross-sectional study performed in 10 European cities. Subjects/Methods: Iron status was defined utilising biochemical indicators. Iron depletion was defined as low serum ferritin (SF < 15 [micro]g/l). Iron deficiency (ID) was defined as high-soluble transferrin receptor (sTfR> 8.5 mg/l) plus iron depletion. Iron deficiency anaemia (IDA) was defined as ID with haemoglobin (Hb) below the WHO cutoff for age and sex: 12.0 g/dl for girls and for boys aged 12.5-14.99 years and 13.0g/dl for boys aged [greater than or equal to] 15 years. Enzyme linked immunosorbent assay was used as analytical method for SF, sTfR and C-reactive protein (CRP). Subjects with indication of inflammation (CRP > 5 mg/l) were excluded from the analyses. A total of 940 adolescents aged 12.5-17.49 years (438 boys and 502 girls) were involved. Results: The percentage of iron depletion was 17.6%, significantly higher in girls (21.0%) compared with boys (13.8%). The overall percentage of ID and IDA was 4.7 and 1.3%, respectively, with no significant differences between boys and girls. A correlation was observed between log (SF) and Hb (r = 0.36, P Conclusions: To ensure adequate iron stores, specific attention should be given to girls at European level to ensure that their dietary intake of iron is adequate. European Journal of Clinical Nutrition (2011) 65, 340-349; doi:10.1038/ejcn.2010.279; published online 19 January 2011 Keywords: anaemia; iron deficiency; adolescent; Europe, Introduction Iron Deficiency (ID) continues to be one of the most prevalent nutritional deficiencies in the world, particularly in developing countries (World Health Organization (WHO), 2003). WHO estimated that ID [...]

The primary route of iron acquisition in vertebrates is the transferrin receptor (TfR) mediated endocytotic pathway, which provides cellular entry to the metal transporter serum transferrin (Tf). Despite extensive research efforts, complete understanding of Tf-TfR interaction mechanism is still lacking owing to the complexity of this system. Electrospray ionization mass spectrometry (ESI MS) is used in this study to monitor the protein/receptor interaction and demonstrate the ability of metal-free Tf to associate with TfR at neutral pH. A set of Tf variants is used in a series of competition and displacement experiments to bracket TfR affinity of apo-Tf at neutral pH (0.2-0.6 [micro]M). Consistent with current models of endosomal iron release from Tf, acidification of the protein solution results in a dramatic change of binding preferences, with apo-Tf becoming a preferred receptor binder. Contrary to the current models implying that the apo-Tf/TfR complex dissociates almost immediately upon exposure to the neutral environment at the cell surface, our data indicate that this complex remains intact. Iron-loaded Tf displaces apo-Tf from TfR, making it available for the next cycle of iron binding, transport and delivery to tissues. However, apo-Tf may still interfere with the cellular uptake of engineered Tf molecules whose TfR affinity is affected by various modifications (e.g., conjugation to cytotoxic molecules). This work also highlights the great potential of ESI MS as a tool capable of providing precise details of complex protein-receptor interactions under conditions that closely mimic the environment in which these encounters occur in physiological systems. metalloprotein | protein interaction doi/ 10.1073/pnas.0914898107

Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-β and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system., Endocytosis is a fundamental process supporting many functions such as nutrient uptake, intracellular signalling (1), morphogenesis during development (2) and defence against pathogens (3). Dysfunctions of the endocytic system lead [...]

Herbison CE, Thorstensen K, Chua AC, Graham RM, Leedman P, Olynyk JK, Trinder D. The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma ceils. Am J Physiol Cell Physiol 297: C1567-C1575, 2009. First published October 14, 2009; doi: 10.1152/ajpcell.00649.2008.--Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFRI and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFRI and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using [.sup.59]Fe-[sup.125]I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. [Fe.sub.2]Tf, [Co.sub.2]Tf, and [Mn.sub.2]Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, [Co.sub.2]Tf and [Mn.sub.2]Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1[alpha] protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFRI knockdown reduced iron uptake by 80% while TFR2 knockdown did not affect uptake. At 5 [micro]M transferrin, iron uptake was not affected by combined TFRI and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15-20% increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells. cobalt; small interfering RNA; chemical hypoxia; metal-transferrin; manganese doi: 10.1152/ajpcell.00649.2008.

Previous research demonstrated that the sympathoadrenal catecholamine norepinephrine could promote the growth of Bordetella bronchiseptica in iron-restricted medium containing serum. In this study, norepinephrine was demonstrated to stimulate growth of this organism in the presence of partially iron-saturated transferrin but not lactoferrin. Although norepinephrine is known to induce transcription of the Bordetella bfeA enterobactin catechol xenosiderophore receptor gene, neither a bfeA mutant nor a bfeR regulator mutant was defective in growth responsiveness to norepinephrine. However, growth of a tonB mutant strain was not enhanced by norepinephrine, indicating that the response to this catecholamine was the result of high-affinity outer membrane transport. The B. bronchiseptica genome encodes a total of 19 known and predicted iron transport receptor genes, none of which, when mutated individually, were found to confer a defect in norepinephrine-mediated growth stimulation in the presence of transferrin. Labeling experiments demonstrated a TonB-dependent increase in cell-associated iron levels when bacteria grown in the presence of [sup.55]Fe-transferrin were exposed to norepinephrine. In addition, TonB was required for maximum levels of cell-associated norepinephrine. Together, these results demonstrate that norepinephrine facilitates B. bronchiseptica iron acquisition from the iron carrier protein transferrin and this process may represent a mechanism by which some bacterial pathogens obtain this essential nutrient in the host environment.

Byline: N. Sheikh, A. Farrukh and A. S. Abbas ABSTRACT Iron and copper, the essential trace elements accomplish their numerous relevant metabolic functions appropriately in the body. Hepcidin, ferroportin-1 and [...]

Despite a lack of transferrin, hypotransferrinemic (Hp) mice demonstrate an accumulation of iron in peripheral organs including the lungs. One potential candidate for such transferrin-independent uptake of iron is divalent metal transporter-1 (DMT1), an established iron transporter. We tested the hypothesis that increased concentrations of iron in the lungs of Hp mice are associated with elevations in DMT1 expression. With the use of inductively coupled plasma emission spectroscopy, measurements of nonheme iron confirmed significantly elevated concentrations in the lung tissue of Hp mice relative to the wild-type mice. Western blot analyses for the expression of two isoforms of DMT1 in the Hp mice relative to the wild-type animals demonstrated an elevation for the isoform that lacks an iron-responsive element (IRE) with significant decrements in the expression of +IRE DMT1. With the use of immunohistochemistry, -IRE DMT1 was localized to both airway epithelial cells and alveolar macrophages in wild-type mice. Staining appeared increased in both types of cells in the Hp mice. Elevated concentrations of both tissue nonheme iron and expression of -IRE DMT1 in the Hp mice were associated with increased quantities of -IRE mRNA. There was no difference between wild-type and homozygotic Hp mice in the amount of mRNA for DMT1 +IRE. We conclude that differences between Hp and wild-type mice in nonheme iron concentrations were accompanied by increases in the expression of -IRE DMTI. Increased expression of -IRE DMT1 in the lungs of the Hp mice could be responsible for elevated concentrations of the metal in these tissues. iron transport; transferrin; membrane transporters

Research has been conducted on mammalian iron-binding proteins lactoferrin and transferrin. The functional and structural differences between these proteins and the factors modulating the interactions in disyline trigger region have been investigated via mutation of Arg 210 to Gly, Glu and Leu in the recombinant N-lobe of human lactoferrin, and the results are reported.

Iron deficiency is the most common micronutrient deficiency worldwide, and transferrin receptor (TfR) level has been identified as an important measure of iron status that is not confounded by inflammation. However, logistical constraints associated with sample collection and processing have limited efforts to measure TfR, particularly at the community level. Standardized filter paper provides a relatively convenient and minimally invasive means for collecting and transporting samples of whole blood from simple finger pricks, and we present results of our validation of an improved method for quantifying TfR in dried blood spots. The method is based on commercially available reagents and uses capillary blood that is applied directly from the finger to filter paper, eliminating the need for premeasurement at the collection site. The blood spot TfR assay is precise and reliable, agrees well with plasma TfR, and can be performed at any facility with a microplate reader and basic laboratory equipment. Concentrations of TfR remain stable for at least 4 wk when blood spots are stored at room temperature, but begin to deteriorate after 3 d of exposure to higher temperatures. The advantages and disadvantages of the blood spot TfR method are discussed, as well as its potential contribution to future field-based studies of iron deficiency. J. Nutr. 132: 3760-3763, 2002. KEY WORDS: * iron deficiency * transferrin receptor * blood specimen collection * immunoassay

Iron deficiency and marginal vitamin A (VA) deficiency frequently coexist and affect billions of people, mostly children and women, worldwide. The effects of these micronutrient deficiencies alone and in combination on hematologic, biochemical and molecular indices of iron and VA status were investigated in a 2 x 2 randomized blocked study conducted in growing male Sprague-Dawley rats. From 3-8 wk of age, rats were fed one of four purified diets that were either adequate or restricted in iron (Fe) and adequate or marginal in VA: [sup.+][Fe.sup.+]VA, 20.3 and 0.367 [micro]/g, respectively, denoted control diet; [sup.-][Fe.sup.+]VA, 3.34 and 0.405 [micro]/g; [sup.+]Fe[VA.sub.m], 22.2 and 0.051 [micro]g/g; or [sup.-]Fe[VA.sub.m], 3.03 and 0.055 [micro]g/g. Weight-matched rats fed adequate micronutrients were included to control for possible confounding effects of Fe deficiency on growth and feed efficiency. Iron restriction reduced (P < 0.05) weight gain, feed efficiency, blood hemoglobin and hematocrit. Plasma and liver iron and plasma transferrin saturation were reduced by ~50%, whereas liver transferrin mRNA and protein and transferrin receptor mRNA were elevated, as was liver ferritin light-chain protein and light-chain mRNA. Liver heavy-chain ferritin mRNA, hemopexin, ceruloplasmin and cellular retinol-binding protein mRNA were not affected by iron or VA restriction. Although marginal VA deficiency did not exacerbate indices of poor iron status during iron deficiency, iron deficiency was associated with lower plasma retinol and elevated liver VA concentrations. These results are consistent with an impaired mobilization of liver retinol during iron deficiency as well as multiple alterations in iron metabolism. J. Nutr. 132: 3607-3615, 2002. KEY WORDS: * iron deficiency * marginal vitamin A deficiency * transferrin * ferritin * rats

Hereditary hemochromatosis (HH) is a disorder of iron metabolism caused by common mutations in the gene HFE. The HFE protein binds to transferrin receptor-1 (TfR1) in competition with transferrin, and in vitro, reduces cellular iron by reducing iron uptake. However, in vivo, HFE is strongly expressed by liver macrophages and intestinal crypt cells, which behave as though they are relatively iron-deficient in HH. These latter observations suggest, paradoxically, that expression of wild-type HFE may lead to iron accumulation in these specialized cell types. Here we show that wild-type HFE protein raises cellular iron by inhibiting iron efflux from the monocyte/macrophage cell line THP-1, and extend these results to macrophages derived from healthy individuals and HH patients. In addition, we find that the HH-associated mutant H41D has lost the ability to inhibit iron release despite binding to TfR1 as well as wild-type HFE. Finally, we show that the ability of HFE to block iron release is not competitively inhibited by transferrin. We conclude that HFE has two mutually exclusive functions, binding to TfR1 in competition with Tf, or inhibition of iron release.

Research has been conducted on gonococcal transferrin binding protein A and TonB. The ability of generated TonB mutants to interact with gonococcal transferrin binding protein A and the effect of characterized TonB-box mutation in gonococcal transferrin binding protein A on gonococcal transferrin binding protein A-TonB interaction have been investigated and discussed.

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis. Caco-2 cells; intestinal iron transport

Enveloped viruses enter cells by binding to their entry receptors and fusing with the membrane at the cell surface or after trafficking through acidic endosomal compartments. Species-specific virus tropism is usually determined by these entry receptors. Because mouse mammary tumor virus (MMTV) is unable to infect Chinese hamster cells, we used phenotypic screening of the T31 mouse/ hamster radiation hybrid panel to map the MMTV cell entry receptor gene and subsequently found that it is transferrin receptor 1. MMTV-resistant human cells that expressed mouse transferrin receptor 1 became susceptible to MMTV infection, and treatment of mouse cells with a monoclonal antibody that down-regulated cell surface expression of the receptor blocked infection. MMTV, like vesicular stomatitis virus, depended on acid pH for infection. MMTV may use transferrin receptor 1, a membrane protein that is endocytosed via clathrin-coated pits and traffics through the acidic endosomes, to rapidly get to a compartment where acid pH triggers the conformational changes in envelope protein required for membrane fusion.

Hereditary hemochromatosis (HH) is a common genetic disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. The vast majority of individuals with HH are homozygous for mutations in the HFE gene. Recently a second transferrin receptor (TFR2) was discovered, and a previously uncharacterized type of hemochromatosis (HH type 3) was identified in humans carrying mutations in the TFR2 gene. To characterize the role for TFR2 in iron homeostasis, we generated mice in which a premature stop codon (Y245X) was introduced by targeted mutagenesis in the murine Tfr2 coding sequence. This mutation is orthologous to the Y250X mutation identified in some patients with HH type 3. The homozygous Tfr[2.sup.Y245X] mutant mice showed profound abnormalities in parameters of iron homeostasis. Even on a standard diet, hepatic iron concentration was several-fold higher in the homozygous Tfr[2.sup.Y245X] mutant mice than in wild-type littermates by 4 weeks of age. The iron deposition in the mutant mice was predominantly hepatocellular and periportal. The mean splenic iron concentration in the homozygous Tfr[2.sup.Y245X] mutant mice was significantly less than that observed in the wild-type mice. The homozygous Tfr[2.sup.Y245X] mutant mice also demonstrated elevated transferrin saturations. There were no significant differences in parameters of erythrocyte production including hemoglobin levels, hematocrits, erythrocyte indices, and reticulocyte counts. Heterozygous Tfr[2.sup.Y245X] mice did not differ in any measured parameter from wild-type mice. This study confirms the important role for TFR2 in iron homeostasis and provides a tool for investigating the excess iron absorption and abnormal iron distribution in iron-overload disorders. iron | liver | gene targeting

Internalization of [beta]-adrenergic receptors ([beta]ARs) occurs by the sequential binding of [beta]-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that [beta]ARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Over-expression of the PIK domain displaces endogenous PI3K from [beta]ARK1 and prevents [beta]ARK1-mediated transiocation of PI3K to activated [[beta]sub.2]ARs. Furthermore, disruption of the [beta]ARK1/P13K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the [[beta].sub.2]AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of P13K to [beta]ARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

Research has been conducted on the recombinant nonglycosylated human serum transferrins. The N-terminal His-tagged recombinant nonglycosylated human serum transferrins has been produced and the results indicate that the C-terminal His tag slows iron release rate from each lobe and that the N-terminal His tag cannot affect this release rate.

Research has been conducted on the human transferrins. The mechanisms of iron release from transferrin in cells and in vitro and of the synergistic iron binding have been investigated and are discussed.

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells. hereditary hemochromatosis; transferrin receptor; patch-clamp capacitance measurements

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both [sup.59]Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells. Key Words: oogenesis; major yolk protein (MYP); yolk platelet; vitellogenin; transferrin; iron.

The protein defective in hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins and associates with [[beta].sub.2]-microglobulin ([[beta].sub.2]M). Its association with [[beta].sub.2]M was previously shown to be necessary for its stability, normal intracellular processing, and cell surface expression in transfected COS cells. Here we use stably transfected Chinese hamster ovary cell lines expressing both HFE and [[beta].sub.2]M or HFE alone to study the effects of [[beta].sub.2]M on the stability and maturation of the HFE protein and on the role of HFE in transferrin receptor 1 (TfR1)-mediated iron uptake. In agreement with prior studies on other cell lines, we found that overexpression of HFE, without overexpressing [[beta].sub.2]M, resulted in a decrease in TfR1-dependent iron uptake and in lower iron levels in the cells, as evidenced by ferritin and TfR1 levels measured at steady state. However, overexpression of both HFE and [[beta].sub.2]M had the reverse effect and resulted in an increase in TfR1-dependent iron uptake and increased iron levels in the cells. The HFE-[[beta].sub.2]M complex did not affect the affinity of TfR1 for transferrin or the internalization rate of transferrin-bound TfR1. Instead, HFE-[[beta].sub.2]M enhanced the rate of recycling of TfR1 and resulted in an increase in the steady-state level of TfR1 at the cell surface of stably transfected cells. We propose that Chinese hamster ovary cells provide a model to explain the effect of the HFE-[[beta].sub.2]M complex in duodenal crypt cells, where the HFE-[[beta].sub.2]M complex appears to facilitate the uptake of transferrin-bound iron to sense the level of body iron stores. Impairment of this process in duodenal crypt cells leads them to be iron poor and to signal the differentiating enterocytes to take up iron excessively after they mature into villus cells in the duodenum of hereditary hemochromatosis patients.

The [mu]2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that [mu]2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of [alpha]-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the [mu] subunit in vitro, and stage-specific assays for endocytosis show that [mu] phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous [mu]2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.

In mammalian cells, internalized receptors such as transferrin (Tfn) receptor are presumed to pass sequentially through early endosomes (EEs) and perinuclear recycling endosomes (REs) before returning to the plasma membrane. Whether passage through RE is obligatory, however, remains unclear. Kinetic analysis of endocytosis in CHO cells suggested that the majority of internalized Tfn bypassed REs returning to the surface from EEs. To determine directly if REs are dispensable for recycling, we studied Tfn recycling in cytoplasts microsurgically created to contain peripheral EEs but to exclude perinuclear REs. The cytoplasts actively internalized and recycled Tfn. Surprisingly, they also exhibited spatially and temporally distinct endosome populations. The first appeared to correspond to EEs, labeling initially with Tfn, being positive for early endosomal antigen 1 (EEA-1) and containing only small amounts of Rab11, an RE marker. The second was EEA-1 negative and with time recruited Rab11, suggesting that cytoplasts assembled functional REs. These results suggest that although perinuclear REs are not essential components of the Tfn recycling pathway, they are dynamic structures which preexist in the peripheral cytoplasm or can be regenerated from EE- and cytosol-derived components such as Rab11.

Research has been conducted on the eukaryotic transferrins, bilobal iron-binding proteins with the binding site in each lobe. The separation of the lobes after the full-length protein expression has been carried out and the results demonstrate the the primary receptor-recognition transferrin site is in its C-lobe.

Research has been conducted on the proteins of the transferrin family. The role of these proteins in the iron homeostasis in vertebrates has been investigated and discussed.

Research has been conducted on the lysine-specific cysteine proteinases (gingipains) of Porphyromonas gingivalis (Kgp). Results demonstrate the ability of the Kgp to bind and degrade hemoglobin, soluble haptoglobin, hemopexin and transferrin present in human serum.

Research reveals that hydroxamate siderophore aerobactin is a 21-kilobase iron trnsport island located downstream of the pheU tRNA gene in Shigella boydii 0-1392. Data indicate that a conserved aerobactin operon contains a P4 prophage-like integrase gene and several insertion sequences, which are localized to different sites within the the genome.

The sequencing and and functional analysis of a cosmid encoding gene in the regulation, biosynthesis, and rhizobactin siderphore 1021 uptake in Sinorhizobium indicate that siderphore biosynthesis and transport genes are not expressed during nitrogenase biosyntheis. However, mutants with insertion elements in these genes induce nitrogen-nodules.

Crystal structure analysis of the N-lobe half-molecule of human transferrin, which mediates iron transport, reveals a highly conserved iron binding site and hydrogen bonding interaction. Data point out that the binding and release of iron is influenced by the dilysine interaction.

Research demonstrates an 82-kilodalton receptor protein from Pasteurella multocida mediating iron uptake from bovine transferrin. Amino acid sequence analysis indicates that the ThpA receptor protein belongs to the TonB-dependent outer membrane receptor family.

Results demonstrate that norepinephrine (NE) positively influences the iron uptake leading to stimulation of bacterial growth under iron-restricted growth conditions as in the presence of iron-binding glycoproteins transferrin and lactoferin. Data indicate that NE-transferrin and NE-lactoferrin complexes are the sole stimulators of growth.

It has been shown the the interactions of Cp(sub.2)TiCl(sub.2) with human serum transferrin (hTF) and that of Ti(sub.2)-hTF with adenosine triphosphate (ATP) may make it possible for transferrin to be a mediator for titanium delivery to tumor cells. Cp(sub.2)TiCl(sub.2), an organometallic cancer agent, is in phase II clinical trials as an anticancer drug. The mechanism of action, however, is not well understood. It appears that titanium transferrin may be recognized by the transferrin receptor and be taken into cancer cells.

Results suggest that PvdS of Pseudomonas aeruginosa belongs to a family of extracytoplasmic alternative sigma factors analogous to eukaryotes. Data reveal that PvdS activates transcription of genes involved in the biosynthesis or the uptake of siderophores.

A study was conducted to analyze the cellular mechanism by which basolateral transferrin produces an increase in apical-to-basolateral iron flux in Caco-2 cells. Insert-grown Caco-2 cells were preincubated in Iscove medium at 37 degrees C. Results indicated that the Ca2+ channel inhibitor SKF96365 inhibited the transferrin-mediated increase in transepithelial iron transport and the secretion of apolipoprotein A.

Iron deficiency in young rats leads to a decrease in brain iron and ferritin concentrations, an increase in transferrin (Tf) concentration, and an increased rate of uptake of iron from the plasma pool. We conducted two experiments to determine whether brain iron, Tf and ferritin respond quickly to iron repletion and to determine whether brain regions respond heterogeneously. Weanling male Sprague-Dawley rats were fed an iron-deficient diet ( KEY WORDS: iron deficiency anemia; transferrin; ferritin; brain; rats

An experiment designed to analyze the function of transferrin receptors and the expression of ferritin genes in the intestinal tract of rats is described. Healthy Wistar rats were given food containing three different iron levels. In situ hybridization was conducted to determine ferritin gene expression while immunohistochemical localization was applied for ferritin gene function. Results reveal greater iron uptake by the small intestine in rats compared with the liver when iron stores were increased.

The effect of transferrin on cartilage neovascularization through promoting endothelial cell migration and invasion was investigated through in vivo assays on chicken egg chorioallantoic membrane. Results have shown that endothelial cell migration was inhibited following induction of polyclonal antibodies directed against transferrin and its receptors indicating that transferrin is an essential factor promoting cell migration in hypertrophic chondrocyte-conditioned culture medium and hypertrophic cartilage in vivo.

Although the absorption of iron through the intestinal epithelia is inversely related to body iron stores, the mechanisms by which the enterocytes sense body iron stores are unknown. Polarized enterocytes have transferrin receptors in their basolateral surface; hence, we tested the hypothesis that the endocytosis of circulating transferrin may be part of the body's iron sensing mechanism. Particularly, we evaluated the contribution of basolateral transferrin to iron content of intestinal cells, and we investigated what factors modulate this contribution. For this purpose, we used the intestinal cell line Caco-2 grown on porous filters. When the cells were simultaneously offered equimolar amounts of iron, from the apical medium as 55Fe-nitrilotriacetate and from the basolateral medium as 59Fe-transferrin, most of the internalized iron came from the basolateral endocytosis of 59Fe-transferrin. Experiments of transferrin binding and internalization showed that holotransferrin and apotransferrin had similar numbers of basolateral receptors. The receptors had associated constants for diferric transferrin, monoferric transferrin, and apotransferrin of 2.69, 1.71 and 0.26 x [10.sup.7] L/mol, respectively. The binding of diferric transferrin or monoferric transferrin to receptors was competitively inhibited by apotransferrin. Caco-2 cells, but not K562 cells, showed inhibition of basolateral, transferrin-mediated iron uptake by apo-transferrin. This inhibition should regulate the net basolateral uptake of iron mediated by the endocytosis of Fe-containing transferrins. We propose that the basolateral endocytosis of transferrin forms part of the system by which intestinal epithelia cells sense plasma iron concentrations. J. Nutr. 126: 2151-2158, 1996. INDEXING KEY WORDS: * Caco-2 cells * iron absorption * enterocyte * transferrin receptor * apotransferrin

Circulating free iron is lethal. Humans have two circulating iron binding proteins to soak up free iron to prevent it from generating toxic quantities of free radicals. These proteins are transferrin, a high-affinity, low-capacity protein (2 atoms of iron per molecule of transferrin) for which there are receptors on the surface of every iron-requiring cell; and ferritin, a lower-affinity, high-capacity protein (maximum of 4500 atoms of iron per molecule of ferritin) for which there are receptors only on the surface of iron-storage cells such as RE (reticulo-endothelial) cells. Iron is trapped inside the ferritin protein shell as harmless [Fe.sub.3]. When there is a high serum level of reduced ascorbic acid, it drives through the pores of the ferritin protein shell to the inside surface, where it converts the [Fe.sub.3] to catalytic [Fe.sub.2], which then leaks out of the pores of the ferritin protein shell and generates billions of free radicals. In normal individuals, per milliliter of serum, there are approximately 300,000 molecules of transferrin per molecule of ferritin. Ferritin protein is an acute phase reactant that sharply rises in the presence of inflammation of any kind, whereas transferrin is a reverse acute phase reactant that falls in the presence of inflammation of any kind. J. Nutr. 126: 1213S-1220S, 1996. INDEXING KEY WORDS: vitamin C, iron, free radicals, ferritin protein, ferritin iron, transferrin

The transferrin-binding protein (TFBP) complex functions as the receptor for the uptake of transferrin in Trypanosoma brucei by a mechanism different from that in mammalian cells. The TFBP complex is comprised of the products of two genes in T. brucei, ESAG 6 and ESAG 7, sited upstream of the variant surface glycoprotein gene in a polycistronic expression site. The complex is a heterodimer that binds one molecule of transferrin with high affinity.

The use of fluorescent transferrin (Tf) and LDL as probes, and confocal microscopy help study the rate and pathway of Tf transport from sorting endosomes of HEp2 human cancer cells. Labeling HEp2 cells to steady state with fluorescein Tf lowers the rate of Tf transport to a level less than that achieved with the use of short filling pulses. Return of Tf through a nonvectorial, retrograde pathway results in excess of Tf in the endosomes.