Structural and functional analyses of chicken liver ferritin.

Characterization of ferritins from different species has provided insight into iron regulation mechanisms and evolutionary relationships. Here, we examined chicken liver ferritin, which comprises only H subunit and has 14.8 µg of Fe/100 µg of protein. The chicken H subunit apo homopolymer showed the same iron uptake rate as bovine H subunit homopolymer expressed with a baculovirus expression system (0.31 and 0.28 mmol of Fe/min per micromole of protein for chicken and bovine H subunit, respectively). Chicken H subunit apo homopolymer showed a significantly higher biotinylated hemin-binding activity than liver holoferritin. Although bovine spleen apoferritin, which has an L (liver or light):H (heart or heavy) subunit ratio of 1:1, also shows a significantly higher biotinylated hemin-binding activity than its holoferritin, these biotinylated hemin-binding activities were markedly lower than those of both chicken holo- and apoferritins. Binding of chicken holo- and apoferritin with biotinylated hemin was strongly inhibited by hemin but not iron-free hemin, protoporphyrin IX, or Zn-protoporphyrin. These findings demonstrate that chicken ferritin comprises only an H subunit, possesses ferroxidase activity as in mammalian ferritin H subunits, and binds heme more strongly than mammalian ferritins.

Short communication: Bovine alpha-casein is a ferritin-binding protein and inhibitory factor of milk ferritin immunoassay.

Commercial bovine milk alpha-casein, but not beta- and kappa-caseins, bound to bovine spleen ferritin, as determined by an immunoassay for ferritin. In contrast, alpha-casein did not bind to apoferritin. The binding of alpha-casein to bovine spleen ferritin was strongly inhibited by increasing ionic strength by the addition of 0.5 M (NH(4))(2)SO(4). The addition of alpha-casein to a known amount of bovine spleen ferritin resulted in significantly lower recovery (78-80%) of added ferritin, although beta- and kappa-caseins showed little inhibitory effect in the ferritin immunoassay. These results indicate that bovine alpha-casein is a specific ferritin-binding protein that may inhibit milk ferritin immunoassay.

Technical note: measurement of ferritin in bovine milk and its clinical significance.

A quantitative ELISA was developed for bovine milk ferritin with an assay limit of 0.16 ng/mL of bovine spleen ferritin. Ferritin-binding activity was detected in bovine milk samples, and this binding activity was inhibited by increasing ionic strength with the addition of 0.5 M (NH4)2SO4. Heat treatment (60 degrees C, 20 min) of bovine milk in the presence of 0.5 M (NH4)2SO4 resulted in a 15 to 58% increase in ferritin concentrations compared with untreated samples. Although the recovery of bovine spleen ferritin added to milk was still low (55 to 90%), even in the presence of increased ionic strength with 0.5 M (NH4)2SO4, recovery was improved by heat treatment at 60 degrees C for 20 min (92 to 95%). Milk ferritin concentrations in 30 milk samples from quarters of 25 cows with mastitis (mean +/- SE: 134.2 +/- 28.7 ng/mL) were significantly higher than those in 17 quarter milk samples from 17 noninfected lactating cows (7.2 +/- 1.2 ng/mL), suggesting that bovine milk contains putative ferritin-binding proteins that inhibit immunoassay for milk ferritin and that bovine milk ferritin is an indicator of IMI.

[Thymolipoma in association with myasthenia gravis; report of a case].

An 18-year-old man was admitted with easy fatigue and muscle weakness. A diagnosis of myasthenia gravis was made. Chest X-ray showed no mediastinal abnormality, however, chest computed tomography (CT) showed a soft tissue mass in the thymus. The patient underwent extended thymectomy and small tumor (2 x 2 x 2 cm) was resected. On histological examination the tumor proved to be a thymolipoma composed of mature adipose elements containing cords and nests of thymic tissue. Symptoms of myasthenia gravis were dramatically improved after the operation. This case is the 12th in the world literature in which a thymolipoma is associated with symptoms of myasthenia gravis.

Iron content of rat serum ferritin.

The serum ferritin concentration was significantly higher in female than in male rats, reflecting higher iron stores in females than in males. The mean iron/protein ratio of serum ferritin was 0.018+/-0.008 (SD) (microg of Fe/microg of protein) in female rats and 0.011+/-0.011 in male rats, being much lower than that of liver ferritin (0.233+/-0.014 in females and 0.227+/-0.020 in males). Iron loading of rats significantly increased serum ferritin concentration, but did not influence the iron content of serum ferritin. These results indicate that rat serum ferritin contains only a small amount of iron independent of body iron stores.

Ferritin and the response to oxidative stress.

Iron is required for normal cell growth and proliferation. However, excess iron is potentially harmful, as it can catalyse the formation of toxic reactive oxygen species (ROS) via Fenton chemistry. For this reason, cells have evolved highly regulated mechanisms for controlling intracellular iron levels. Chief among these is the sequestration of iron in ferritin. Ferritin is a 24 subunit protein composed of two subunit types, termed H and L. The ferritin H subunit has a potent ferroxidase activity that catalyses the oxidation of ferrous iron, whereas ferritin L plays a role in iron nucleation and protein stability. In the present study we report that increased synthesis of both subunits of ferritin occurs in HeLa cells exposed to oxidative stress. An increase in the activity of iron responsive element binding proteins in response to oxidative stress was also observed. However, this activation was transient, allowing ferritin protein induction to subsequently proceed. To assess whether ferritin induction reduced the accumulation of ROS, and to test the relative contribution of ferritin H and L subunits in this process, we prepared stable transfectants that overexpressed either ferritin H or ferritin L cDNA under control of a tetracycline-responsive promoter. We observed that overexpression of either ferritin H or ferritin L reduced the accumulation of ROS in response to oxidant challenge.

Biochemical properties of canine serum ferritin: iron content and nonbinding to concanavalin A.

A sandwich enzyme-linked immunosorbent assay using H-subunit-rich canine heart ferritin as a standard has been developed for measuring canine serum ferritin which is H-subunit-rich. Serum ferritin concentrations in 51 normal dogs ranged from 143 to 1766 ng ml(-1), with a mean value of 479 +/- 286 (SD) ng ml(-1). Serum ferritin iron concentrations as determined by an immunoprecipitation technique ranged from 30.4 to 115.9 ng ml(-1) in 15 normal dogs with serum ferritin protein levels of 298 to 959 ng ml(-1). There was a significant linear correlation between the serum ferritin iron and protein levels (r=0.9441, P<0.001), and the mean iron/protein ratio of serum ferritin was 0.112 +/- 0.017. When canine sera were incubated with concanavalin A-Sepharose 4B, we observed the apparent binding of serum ferritin to concanavalin A. However, ferritin obtained by heat-treating the sera at pH 4.8 to remove the ferritin-binding proteins did not bind to the lectin. These results suggest that canine serum ferritin contains a considerable amount of iron but no concanavalin A-binding G subunit present in human serum ferritin.

Adenovirus E1A blocks oxidant-dependent ferritin induction and sensitizes cells to pro-oxidant cytotoxicity.

Ferritin is a protein that oxidizes and sequesters intracellular iron in a mineral core. We have reported that the E1A oncogene selectively represses ferritin H transcription, resulting in reduced levels of the ferritin H protein. Here we demonstrate that cells respond to pro-oxidant challenge by inducing ferritin mRNA and protein, and that this response is completely blocked by E1A. Concordantly, E1A sensitized cells to the cytotoxic effects of oxidative stress and enhanced the accumulation of reactive oxygen species in response to pro-oxidant challenge. These results demonstrate that expression of E1A impedes the cellular response to oxidative stress, including the induction of ferritin.

Changes in iron and ferritin in anemic calves infected with Theileria sergenti.

Changes in iron and ferritin in calves infected with Theileria sergenti were investigated to elucidate iron metabolism in animals with extravascular hemolytic anemia. During severe anemia, serum iron was remarkably elevated while the total iron-binding capacity remained relatively unchanged or decreased slightly in the infected calves, resulting in elevated transferrin saturation. The serum ferritin concentration gradually increased with the progress of anemia. The erythrocyte ferritin content drastically increased when mean corpuscular volume was elevated. The concentration of non-heme iron and ferritin in the liver, spleen, and bone marrow of the infected calves was markedly higher than that in the respective tissues of the control animals. In particular, the liver of the anemic calves was found to contain 23 and 35 times as much non-heme iron and ferritin, respectively, as that of the non-anemic healthy cattle. The liver type (L) to heart type (H) subunit ratio of liver ferritin was significantly higher in the protozoa-infected than in the non-infected cattle. On the other hand, the L/H ratio of marrow ferritin was significantly reduced by the anemia. These results indicate that the anemic calves infected with T. sergenti apparently present symptoms of iron overload.

[Efficacy of F-18 fluorodeoxyglucose positron emission tomography (FDG-PET) scans in diagnosis of pulmonary nodules].

To evaluate the value of F-18 fluorodeoxyglucose positron emission tomography (FDG-PET) scans, we performed FDG-PET scans in 23 patients with indeterminate pulmonary nodules less than 3 cm in size and analyzed these scans qualitatively and semiquantitatively. Histologic specimens were obtained by thoracoscopic excisional biopsy in 16 patients, CT-guided needle aspiration cytology in three, and bronchoscopic brushing cytology in four. Pathological diagnoses were lung cancer in 16 patients, benign inflammation in six, and malignant lymphoma in one. Sensitivity, specificity and accuracy of the FDG-PET scans were 88% (15/17), 67% (4/6) and 83% (19/23), respectively. There were two false-positive cases (organizing pneumonia and cryptococcosis) and two false-negative ones (slow-growing adenocarcinoma and malignant lymphoma). Although a few false-positive cases of granulomatous disease were yielded, the FDG-PET scans were highly sensitive in the detection of lung cancer. We conclude that the FDG-PET scanning in a useful diagnostic imaging modailty in the management of indeterminate pulmonary nodules.

High levels of ferritin and its iron in fetal bovine serum.

In 13 lots of the commercial fetal bovine sera, the ferritin levels ranged between 0.8 and 6.0 micrograms/ml. The serum ferritin iron concentration as measured by a quantitative immunoprecipitation technique ranged from 0.16 to 0.96 microgram/ml, and the iron content of ferritin was about 20% regardless of its protein concentration in sera. The percentage of ferritin iron to total serum iron ranged from 8.8 to 28.5%, and correlated significantly with ferritin concentration (r = 0.9368, P < 0.001). No significant proportion of the ferritin in fetal serum bound to concanavalin A. Immunoblotting showed that the molecular weights of L(iver)- and H(eart)-type subunits of fetal serum ferritin were identical to those of L and H subunits of adult bovine spleen ferritin (L:21kDa, H:18kDa), respectively, and that the L subunit predominated in the serum protein. Serum transferrin level was relatively constant (1.8-2.2 mg/ml), whereas transferrin saturation varied from 54.8 to 91.7%. There was a significant correlation between serum ferritin concentration and transferrin saturation (r = 0.8864, P < 0.001). These findings demonstrate that the bovine fetuses have the elevated iron stores.

Sequencing of cDNA clones that encode bovine ferritin H and L chains.

The molecular weight of the liver-type subunit (L) of bovine ferritin is much larger than that of the heart-type subunit (H) as determined by SDS-PAGE (L, 20.5 kDa; H, 18.4 kDa). The migration of these two subunits on SDS-PAGE gels, relative to each other, is opposite to that reported for ferritin L and H subunits in other mammalian species (L, 19 kDa; H, 21 kDa). To determine the cause of this anomaly, full-length cDNA clones of the bovine L and H chains were isolated from a bovine spleen gamma gt11 cDNA library and sequenced. The amino acid sequences of the L and H chains of bovine ferritin, deduced from their cDNA sequences, contained open reading frames coding for 174 and 180 amino acid residues with calculated molecular weights of 19,856 and 20,920 Da, respectively. The deduced amino acid sequence of the L chain shows 86%, 84%, 87%, 83% and 83% homology with the amino acid sequences of horse, human, rabbit, rat and mouse L chains, respectively. The H chain displays a higher homology with the human, rat and mouse H chains (91%, 92% and 93%, respectively). In addition, the bovine L chain did not contain the extra octapeptide present in rodent L chains, and bovine, L and H chains did not react with concanavalin A. The bovine L and H chains expressed using a baculovirus expression system showed almost the same mobilities as those of bovine spleen ferritin, respectively, by SDS-PAGE. These results suggest that the much slower mobility of the bovine L chain compared with other mammalian L chains on SDS-PAGE cannot be attributed to insertion(s) of amino acid(s) or peptide(s) into the L chain, to the deletion(s) of them of it or to the addition of carbohydrate chains(s) but may result from significant differences in the binding affinity of SDS for bovine ferritin L chains.

[The ethanol elimination pharmacokinetics--the effects of genotypes of ALDH2 and CYP2E1 on the ethanol metabolism].

The effects of the genotypes of CYP2E1, ALDH2, ADH upon the blood ethanol and acetaldehyde levels were investigated. The predicting 95% confidence bounds determined on regression analysis of the data suggested that after venous injection of ethanol, the blood ethanol and acetaldehyde concentrations in a volunteer normal homozygous for ALDH2 (ALDH2*1/1) were significantly lower than that heterozygous (ALDH2*1/2). And the blood ethanol and acetaldehyde concentrations in a volunteer with C2 allele (C1/C2) were significantly lower than that in (C1/1). However, there were no significant differences in the blood ethanol and acetaldehyde concentrations between volunteers with ADH2*1/1 and ALDH2*1/2. It is possible that the ALDH2*1 and C2 alleles may correspond to the lower blood ethanol and acetaldehyde concentrations after intravenous administrations of 0.2 g /kg of ethanol.

Immunological cross-reaction between lactoferrin and transferrin.

Hardly any or only a weak immunological cross-reaction was found between native lactoferrin (Lf) and transferrin (Tf). However, when these iron-binding proteins were denatured with sodium dodecyl sulfate and dithiothreitol, a definite immunological cross-reaction was detected between them. These results indicate that although Lf and Tf are immunologically quite different from each other in their native forms, they have the common antigenic determinant(s) in their unfolded forms.

Identification of bovine serum transferrin phenotypes by polyacrylamide gel isoelectric focusing (PAGIEF).

Each of four common homozygous bovine serum transferrins Tf A, Tf D1, Tf D2, and Tf E gave only two main bands in polyacrylamide gel isoelectric focusing (PAGIEF). This is considered to be due to the co-migration of the main components 2a and 3a with the main components 2b and 3b, respectively, in PAGIEF. Ten phenotypes, which are controlled by four alleles TfA, TfD1, TfD2, and TfE, were distinguishable from each other in PAGIEF.

[The genotype of CYP2E1 and liver-derived serum enzymes].

We determined the genotypes of the CYP2E1 loci of Japanese alcoholics with or without liver dysfunction to investigate the relationship between CYP2E1 and the susceptibility to alcoholic liver-injury. In the alcoholics (DSM-III-R) with a liver dysfunction, there was a significant relationship between the serum concentration of a liver-derived serum enzyme LAP and the C2 allele of CYP2E1.

[Relationship between alcoholism and genotypes of acetaldehyde-metabolizing enzymes].

The genotypes of the CYP2E1 and ALDH2 loci in alcoholic and non-alcoholic (healthy) Japanese were investigated to examine the relationship between the polymorphisms of CYP2E1 (C1/C2) and ALDH2 (ALDH2*1/ALDH2*2), and the susceptibility to alcoholism. There was no significant difference in C2 gene frequency between alcoholics (0.20) and non-alcoholics (0.19), while there was a significant difference in ALDH2 allele frequency, suggesting that the C2 allele of CYP2E1 may have nothing to the risk of developing alcoholism in Japanese, whereas the ALDH2*1 allele may influence drinking behavior and the development of alcoholism.

[Contribution of the ALDH2 and CYP2E1 genes to flushing response and drinking patterns in Japanese healthy volunteers].

Relationship between genotypes of the loci for ALDH2 and CYP2E1 and the flushing response as well as drinking pattern were investigated among 31 Japanese healthy persons. In 14 persons who showed flushing symptom and whose ALDH2 genotype was heterozygote (ALDH2*1/ALDH2*2), 6 persons whose CYP2E1 genotype was mutant homozygote or heterozygote (C2/C2 or C1/C2), reported to drink more frequently and their alcohol consumption was higher than that of C1/C1 carriers. However, 2 subjects possessing C2 allele in 3 persons carrying ALDH2 homozygous genotype showed very low alcohol consumption. It is concluded that a person heterozygous for ALDH2 and either mutant homozygous or heterozygous for CYP2E1 may become able to drink more alcohol, if his drinking pattern grows more frequent.

Fibrinogen as a ferritin-binding protein in horse plasma.

Lower apparent concentrations of ferritin were observed in horse plasma than in serum using the enzyme-linked immunosorbent assay (ELISA). However, the ferritin concentrations in plasma and serum were increased to the same level on heating the samples at 75 degrees C for 15 min. These results suggest that horse plasma has specific ferritin-binding protein(s) which inhibit(s) the ferritin assay. The apparent ferritin concentrations in horse serum were markedly decreased by adding horse fibrinogen to the serum. It was also found that fibrinogen bound to spleen ferritin and inhibited the immunoassay of this protein. From these results, it was concluded that horse fibrinogen is one of the ferritin-binding proteins which inhibit the immunoassay of horse ferritin.

Inhibitory effects of horse serum on immunoassay of horse ferritin.

The effects of horse serum on the immunoassay of horse ferritin were investigated using two sandwich enzyme-linked immunosorbent assay (ELISA) systems. In System A, affinity-purified antibody to horse spleen ferritin and its conjugate with alkaline phosphatase were used as the first and second antibodies, respectively. In System B, whole antiserum and its conjugate with the enzyme were used. The recoveries of horse spleen ferritin added to horse sera were very low in either system (50-71% in System A; 42-79% in System B). However, heat treatment of the sera at 75 degrees C for 15 min improved the recoveries (90-96%) in System A, whereas the recoveries in System B were not sufficiently improved by the same treatment (75-83%). The apparent concentrations of ferritin in adult and newborn horse sera increased after heat treatment of the samples. From these results, it is concluded that horse serum contains the heat-unstable substance(s) which inhibits the immunoassay of horse ferritin.