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PubMed2000

Transferrin-bound and transferrin free iron uptake by cultured rat astrocytes.

Previously we had demonstrated the presence of transferrin receptor (TfR) on the plasma membrane of cultured rat cortical astrocytes. In this study, we investigated the roles of TfR in transferrin-bound iron (Tf-Fe) as well as transferrin-free iron (Fe II) uptake by the cells. The cultured rat astrocytes were incubated with 1 microM of double-labelled transferrin (125I-Tf-59Fe) in serum- free DMEM F12 medium or 59Fe II in isotonic sucrose solution at 37 degrees C or 4 degrees C for varying times. The cellular Tf-Fe, Tf and Fe II uptake was analyzed by measuring the intracellular radioactivity with gamma counter. The result showed that Tf-Fe uptake kept increasing in a linear manner at least in the first 30-min. In contrast to Tf-Fe uptake, the internalization of Tf into the cells was rapid initially but then slowed to a plateau level after 10 min. of incubation. The addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake. Pre-treated cells with trypsin inhibited significantly the cellular uptake of Tf-Fe as well as Tf. These findings suggested that Tf-Fe transport across the membrane of astrocytes is mediated by Tf-TfR endocytosis. The results of transferrin-free iron uptake indicated that the cultured rat cortical astrocytes had the capacity to acquire Fe II. The highest uptake of Fe II occurred at pH 6.5. The Fe II uptake was time and temperature dependent, iron concentration saturable, inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+ and not significantly affected by phenylarsine oxide treatment. These characteristics of Fe II uptake by the cultured astrocytes suggested that Fe II uptake is not mediated by TfR and implied that a carrier-mediated iron transport system might be present on the membrane of the cultured cells.

Animals↗
PubMed1990

Identification of the NADH-binding subunit of NADH-ubiquinone oxidoreductase of Paracoccus denitrificans.

The NADH dehydrogenase complex isolated from Paracoccus denitrificans is composed of approximately 10 unlike polypeptides and contains noncovalently bound FMN, non-heme iron, and acid-labile sulfide [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311]. When the Paracoccus NADH dehydrogenase complex was irradiated by UV light in the presence of [adenylate-32P]NAD, radioactivity was incorporated exclusively into one of three polypeptides of Mr approximately 50,000. Similar results were obtained when [adenylate-32P]NADH was used. The labeling of the Mr 50,000 polypeptide was diminished when UV irradiation of the enzyme with [adenylate-32P]NAD was performed in the presence of NADH, but not in the presence of NADP(H). The labeled polypeptide was isolated by preparative sodium dodecyl sulfate gel electrophoresis and was shown to cross-react with antiserum to the NADH-binding subunit (Mr = 51,000) of bovine NADH-ubiquinone oxidoreductase. Its amino acid composition was also very similar to that of the bovine NADH-binding subunit. These chemical and immunological results indicate that the Mr 50,000 polypeptide is an NADH-binding subunit of the Paracoccus NADH dehydrogenase complex.

Animals↗
PubMed1991

Roles of ATP and NADPH in formation of the fe-s cluster of spinach ferredoxin.

Ferredoxin (Fd) in higher plants is encoded by a nuclear gene, synthesized in the cytoplasm as a larger precursor, and imported into the chloroplast, where it is proteolytically processed, and assembled with the [2Fe-2S] cluster. The final step in the biosynthetic pathway of Fd can be analyzed by a reconstitution system composed of isolated chloroplasts and [(35)S]cysteine, in which [(35)S]sulfide and iron are incorporated into Fd to build up the (35)S-labeled Fe-S cluster. Although a lysed chloroplast system shows obligate requirements for ATP and NADPH, in vitro chemical reconstitution of the Fe-S cluster is generally thought to be energy-independent. The present study investigated whether ATP and NADPH in the chloroplast system of spinach (Spinacia oleracea) are involved in the supply of [(35)S]sulfide or iron, or in Fe-S cluster formation itself. [(35)S]Sulfide was liberated from [(35)S] cysteine in an NADPH-dependent manner, whereas ATP was not necessary for this process. This desulfhydration of [(35)S]cysteine occurred before the formation of the (35)S-labeled Fe-S cluster, and the amount of radioactivity in [(35)S]sulfide was greater than that in (35)S-labeled holo-Fd by a factor of more than 20. Addition of nonradioactive sulfide (Na(2)S) inhibited competitively formation of the (35)S-labeled Fe-S cluster along with the addition of nonradioactive cysteine, indicating that some of the inorganic sulfide released from cysteine is incorporated into the Fe-S cluster of Fd. ATP hydrolysis was not involved in the production of inorganic sulfide or in the supply of iron for assembly into the Fe-S cluster. However, ATP-dependent Fe-S cluster formation was observed even in the presence of sufficient amounts of [(35)S]sulfide and iron. These results suggest a novel type of ATP-dependent in vivo Fe-S cluster formation that is distinct from in vitro chemical reconstitution. The implications of these results for the possible mechanisms of ATP-dependent Fe-S cluster formation are discussed.

Journal Article↗
PubMed1984

Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors.

Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites.

Animals↗
PubMed1994

Photoaffinity labeling of cytochrome P450 2B4: capture of active site heme ligands by a photocarbene.

Spiro[adamantane-2,2'-diazirine], which produces adamantyl carbene upon photolysis, binds tightly to P450 2B4 (KS = 3.2 microM), giving a normal substrate binding difference spectrum. Irradiation of 2-[3H]adamantane diazirine at 365 nm in the presence of native, ferric P450 2B4 resulted in first-order photolysis (t1/2 = 1.8 min). The main product was 2-[3H]adamantanol, with about 6% of the radioactivity covalently bound to P450 2B4. With the ferrous carbonyl form of P450 2B4, 2-adamantanol production decreased and protein labeling increased to 12%. When ferric cyanide 2B4 was used, 2-adamantanecarbonitrile was formed in addition to 2-adamantanol. The nitrile appears to have resulted from capture of the iron-bound cyanide ligand by the carbene. The use of multiple cycles of photolysis increased the percentage of protein labeling to 76%. Photolabeling was inhibited by known 2B4 substrates and inhibitors. Also, N-demethylation of benzphetamine and generation of a substrate binding difference spectrum by benzphetamine were both inhibited stoichiometrically with the fraction of radiolabeled protein. The labeled protein was permanently converted to the high-spin state, as indicated by the characteristic change in the absorbance spectrum, demonstrating irreversible occupation of the substrate binding site by the adamantyl residue. Mild acid hydrolysis of radiolabeled 2B4 at the five Asp-Pro bonds generated a 2-kDa peptide which carried 78% of the radioactivity. These results are interpreted as the result of the active site carbene reacting by three competing pathways: capture of the heme sixth ligand to yield either 2-adamantanol or 2-adamantanecarbonitrile, capture of an unbound active site water molecule to yield adamantanol, and covalent attachment to a protein residue. Thus, the P450 2B4 active site appears to contain at least one unbound water molecule in addition to the heme aquo sixth ligand, even when substrate is present.

Adamantane↗
PubMed1987

Receptor-mediated transcytosis of transferrin across the blood-brain barrier.

The perfusion of rat brain with 125I-transferrin resulted in a receptor-mediated uptake of transferrin into the endothelium of the blood-brain barrier followed by its detection in the brain. During a pulse-chase experiment, 125I-transferrin accumulated in the endothelial cells during the pulse, with a decrease of this intraendothelial radioactivity during the chase associated with a concomitant increase in the nonvascular elements of the brain. The receptor-mediated movement of transferrin across the blood-brain barrier suggests that the brain may derive its iron through the transcytosis of iron-loaded transferrin across the brain microvasculature. We discuss the likelihood that aluminum and other potentially toxic heavy metals, which also bind tightly to transferrin, may enter the brain by this pathway. We also discuss the possibility that other large molecules including neuroactive peptides and neurotrophic viruses may enter the brain through a similar receptor-mediated, vesicular transcytotic route.

Animals↗
PubMed2006

Equilibrium isotherm modeling of cesium adsorption onto magnetic materials.

The present work investigates the adsorptive interactions of Cs ions with natural magnetite and synthesized iron ferrite in aqueous medium. The applied adsorbents were characterized by FTIR and DTA/TGA analyses. Batch adsorption studies were performed to evaluate the influences of various experimental parameters like initial pH, contact time and initial concentration on the removal of Cs. The adsorption is strongly dependent on pH of the medium where the removal efficiency increases as the pH turns to alkaline range. The process was very fast initially and maximum adsorption was attained within 60 min of contact. The adsorption process follows a pseudo-second order kinetics with rate constant amounted to 76.83 x 10(4) and 18.75 x 10(4)g mg(-1)h(-1) with ferrite and magnetite. The presence of interfering cations seriously decreases the extent of Cs adsorption. The equilibrium data of Cs adsorption on both adsorbents were analyzed using the Freundlich, Langmuir, Temkin, Dubinin-Radushkevich and Redlich-Peterson isotherm models. The different isotherms constants were determined from the linearzed form of their equations and used to characterize Cs distribution on adsorbent surfaces and provide adopted information about the affinity of the adsorbents towards Cs ions. The values of Langmuir separation factor indicate a favorable Cs adsorption. The apparent free energies from the Dubinin-Radushkevich are 32.29 and 27.51 kJ mol(-1) for Cs adsorption onto iron ferrite and magnetite, respectively.

Adsorption↗
PubMed1986

Erythrocyte enzymes in polycythemia vera: a comparison to erythrocyte enzyme activities of patients with iron deficiency anemia.

Fifteen cytoplasmic erythrocyte enzyme activities were determined in patients with polycythemia vera (PV), iron deficiency anemia (IDA), and a group of healthy volunteers. Among the PV patients, the erythrocyte enzyme activities were compared between 2 groups: patients treated solely with phlebotomy and patients treated with phlebotomy, Myleran (busulfan) and/or radioactive phosphorus 32P. Significant reduction in glutathione reductase activity was found in the PV group of patients. This activity was normalized by the addition of flavin adenine dinucleotide. In contrast to previous reports, no other enzyme activity was found to be significantly reduced. The activities of the enzymes known to be age-dependent were significantly elevated in all the groups tested except for phosphofructokinase and 3-phosphoglycerate mutase. The former was not elevated in any of the groups studied, while the latter was elevated only in the group of patients treated with Myleran and/or 32P. It was concluded that glutathione reductase (GR) deficiency is the only acquired enzyme deficiency in our group of PV patients and that radiation and chemotherapy did not induce further reduction in the activities of any of the enzymes tested. The possible involvement of GR deficiency in the etiology of the red cell life span shortening was discussed.

Anemia, Hypochromic↗
PubMed2003

[A novel method for sentinel lymph node mapping using magnetite].

The detection rate of sentinel lymph nodes using isosulfan blue dye is too low for clinical use. Although exposure to radioactivity is reportedly minimal, special procedures are nonetheless required when a radioactive isotope is used as a tracer. Therefore, to eliminate the need for a radioactive tracer and to obtain a better detection rate than is currently obtained with isosulfan blue dye, we have developed a novel method that employs magnetite as the tracer. The tracer employed is ferumoxides, a colloidal superparamagnetic iron oxide of non-stoichiometric magnetite. The magnetic force within the lymph nodes was measured using a highly sensitive hand-held magnetometer ex vivo. The rate of detection of sentinel lymph nodes was 82% in non-small cell lung cancer. Intraoperative sentinel lymph node mapping using ferumoxides and a highly sensitive magnetometer is a safe, accurate, and sensitive way to detect sentinel lymph nodes. To detect sentinel lymph nodes in vivo, the magnetometer should be sterilized and the sensitivity of the magnetometer should be higher than that we used. Therefore now we are developing a more sensitive and sterilizable magnetometer.

Contrast Media↗
PubMed1984

Correlation between the behavior of magnetic iron oxide particles in the lungs of rabbits and phagocytosis.

Five New Zealand white male rabbits were exposed (30 min; 300 mg/m3) to a submicrometric magnetic iron oxide aerosol (gamma-Fe2O3) produced by burning iron pentacarbonyl in a reducing atmosphere. After aerosol inhalation, an external magnetic field was applied to the rabbits to magnetize and align the ferrimagnetic particles within their lungs. After removal of the external field, a remanent magnetic field was detectable at the body surface. Using a flux-gate magnetometer probe in an enclosure shielded against external magnetic noise, the peak remanent field after magnetization was measured periodically during the next 6 weeks. After each magnetization, the strength of the remanent field decayed rapidly with time (relaxation). The mechanism responsible is particle rotation caused by tissue, cell, organelle, or Brownian movement. The rate of relaxation changed with time after particle inhalation, especially during the first day; changes in the relaxation rate correlated with an estimate of in situ particle phagocytosis during that time. Analysis of pulmonary lavage fluid from 15 rabbits into which radioactive gold-198 had been intratracheally instilled showed that, at 1 hr after instillation, 27% of the gold had been phagocytized, whereas at 16 hr 91% had been ingested. The strength of the magnetic field immediately after each magnetization (that is, before relaxation) was used to estimate the amount of iron oxide in the lungs. At 1 day after exposure, 96.8 +/- 8.8% (mean +/- standard error) of the initial dust was still present; at 10 days, 67.9 +/- 16.2%; and at 40 days, 16.0 +/- 4.6%. It is concluded that ferrimagnetic particles can serve as an easily measured, long-lasting marker that can be used for noninvasive studies of clearance and of particle phagocytosis and as a probe for intracellular processes such as organelle motion.

Aerosols↗