Real Time Detection of Reactions Between Radicals of Lycopene and Tocopherol Homologues

Laser flash photolysis of lycopene in homogeneous chloroform solution together with tocopherol homolopes results in rapid formation of the lycopene radical cation and slower formation of tocopheroxyl radicals. Time-resolved detection by absorption spectroscopy of decay of the lycopene radical cation, of formation of the tocopheroxyl radicals, and of bleaching of lycopene has shown that a-tocopherol is able to reduce the lycopene radical cation and thereby partially regenerate lycopene on a ms timescale. In contrast, lycopene is able to reduce the δ-tocopheroxyl radical, whereas an equilibrium exists between the lycopene radical cation and β- or γ-tocopherol. The relative stability of these antioxidant radicals is hence: a-tocopheroxyl > lycopene radical cation ≈ β-tocopheroxyl - γ-tocopheroxyl > S-toco-pheroxyl.     

Laser photobleaching of human serum: application to fluorescence immunoassays

Background fluorescence from serum chromophores is substantially reduced by a laser photobleaching method. Human and bovine serum samples were illuminated with 337-nm light from a pulsed N2 laser for a short period of time. The serum emission in the region of 440 to 550 nm was reduced by an order of magnitude with no evident damage to serum proteins as judged by the unchanged activity of alkaline phosphatase and aspartate aminotransferase.     

Simultaneous production of immunoaffinity membranes

We simultaneously separated antibodies for transferrin, the third component of complement (C3), haptoglobin and transthyretin by multi-sample non-denaturing two-dimensional electrophoresis (2-DE), transferred them to a polyvinylidene difluoride (PVDF) membrane and then stained them using direct blue 71 to obtain membrane-immobilized antibodies. The antigens, transferrin, C3, haptoglobin and transthyretin were specifically bound to the membrane-immobilized antibodies and were eluted only after rinsing the membrane with acid solution. The antigens specifically bound to the membrane-immobilized antibodies were separated by SDS-PAGE and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, transferrin and transthyretin were trapped and eluted by each membrane-immobilized antibody and detected by MALDI-TOF MS directly without separations. Using membrane-immobilized anti-transferrin antibody, transferrin in flowing blood was directly trapped and analyzed. The results indicated that membrane-immobilized antibodies are simultaneously produced, and that the immunoaffinity membranes can capture specific substances in flowing fluids.     

The bacterial receptor protein, transferrin-binding protein B, does not independently facilitate the release of metal ion from human transferrin.

Pathogenic Gram-negative bacteria of the Pasteurellaceae and Neisseriaceae acquire iron for growth from host transferrin through the action of specific surface receptors. Iron is removed from transferrin by the receptor at the cell surface and is transported across the outer membrane to the periplasm. A periplasmic binding protein-dependent pathway subsequently transports iron into the cell. The transferrin receptor is composed of a largely surface-exposed lipoprotein, transferrin binding protein B, and a TonB-dependent integral outer membrane protein, transferrin binding protein A. To examine the role of transferrin binding protein B in the iron removal process, complexes of recombinant transferrin binding protein B and transferrin were prepared and compared with transferrin in metal-binding and -removal experiments. A polyhistidine-tagged form of recombinant transferrin binding protein B was able to purify a complex with transferrin that was largely monodisperse by dynamic light scattering analysis. Gallium was used instead of iron in the metal-binding studies, since it resulted in increased stability of recombinant transferrin binding protein B in the complex. Difference absorption spectra were used to monitor removal of gallium by nitrilotriacetic acid. Kinetic and equilibrium binding studies indicated that transferrin binds gallium more tightly in the presence of transferrin binding protein B. Thus, transferrin binding protein B does not facilitate metal ion removal and additional components are required for this process.     

Monoclonal antibodies against pig transierrin. Blocking and binding activity

Monoclonal mouse anti-pig transferrin antibodies PTF-01, PTF-02 and PTF-03 and anti-human transferrin antibody HTF-14 detect transferrin coupled with Sepharose particles in an indirect immunofluorescence test. Only the PTF-03 antibody can be used for immunofluorescence detection of pig transferrin bound to specific receptors on the plasma membrane. The binding of iodinated pig transferrin to PK cells was studied. It could be blocked by nonlabelled transferrin in excess, by pig serum or by anti-pig transferrin monoclonal antibodies. PTF-03 expressed the lowest blocking activity among the antibodies tested.     

Studies on the forms of iron-transferrin released from rabbit reticulocytes

Measurement of the distribution of the four species of transferrin, viz, apotransferrin, diferric transferrin and the two monoferric transferrin, before and after incubation of iron-rich rabbit transferrin with rabbit reticulocytes showed that not all transferrin released from the cells were in the form of apotransferrin. Instead, a mixture of all four species of the protein was released with apotransferrin and C-terminal monoferric transferrin being the major fractions. The buffer solution containing 125I-labelled transferrin showed a continuous gain in percentages in apotransferrin and C-terminal monoferric transferrin after each incubation with reticulocytes. The N-terminal monoferric transferrin, however, remained unchanged suggesting that in the process of transferrin uptake by cells, the diferric transferrin releases its iron from the acid-labile site at N-domain first before the other iron from the acid-stable site.     

Terpenoid Induction in Sweet Potato Roots by Cyclic-3′, 5′-Adenosine Monophosphate

The bar-headed goose, a specialized high-altitude species, has a capacity for high oxygen uptake from a hypoxic environment. It thus has a higher oxygen affinity than other bird species of lower-altitude environments. Oxygen affinity is determined by molecular structures and genetic mutations of hemoglobin (Hb), which can also influence the coordinating structures and dynamics of oxygen-Hb. To explore the structural differences in Hbs as between high and low altitude species, photolysis dynamic parameters, including quantum yield, enthalpy, and conformational volume changes in carboxy-Hbs (HbCO) for the bar-headed goose and low altitude counterparts (the Chinese goose and chicken) were investigated by the laser pumping-probing technique and photoacoustic calorimetry. Comparing the photolysis results for HbCO of the three species, the enthalpy and conformational volume changes of the bar-headed goose were much smaller than those of the others, although the quantum yields of all three species are similar. To explain the possible mechanisms of these differences, modifications of salt bridges and key residue mutations at the α β subunit interfaces of the proteins are described and discussed briefly.     

NADH diferric transferrin reductase in liver plasma membrane

Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.     

Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits

Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K⁺ depletion of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin receptor complex by clathrin-independent pathway.     

Diagnosis of congenital disorders of glycosylation type-I using protein chip technology

A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics.     

Preferential utilization in vitro of iron bound to diferric transferrin by rabbit reticulocytes.

59Fe uptake by rabbit reticulocytes from human transferrin-bound iron was studied by using transferrin solutions (35, 50, 65, 80 and 100% saturated with iron) whose only common characteristic was their content of diferric transferrin. During the early incubation period, 59Fe uptake from each preparation by reticulocytes was identical despite wide variations in amounts of total transferrin, total iron, monoferric transferrin and apotransferrin in solution. During the later phase of incubation, rate of uptake declined and was proportional to each solution's monoferric transferrin content. Uptake was also studied in a comparative experiment which used two identical, partially saturated transferrin preparations, one uniformly 59Fe-labelled and the other tracer-labelled with [59Fe]diferric transferrin. In both experiments, iron uptake by reticulocytes corresponded to utilization of a ferric ion from diferric transferrin before utilization of iron from monoferric transferrin.     

In vivo behaviour of rat transferrin bearing a hybrid glycan and its interaction with macrophages.

Production of rat transferrin containing a single hybrid glycan was induced by treating rats with swainsonine, an inhibitor of alpha-mannosidase II. The principal component of this variant transferrin containing one sialic acid residue per mole of protein was separated from other forms of transferrin by anion-exchange chromatography, followed by lectin affinity chromatography. Transferrin bearing the hybrid glycan was degraded in vivo with a half-life of 14 h as compared with 40 h for transferrin containing a standard diantennary glycan. By using 125I-labelled tyramine-cellobiose, a label whose discharge from lysosomes is strongly retarded, organs rich in reticuloendothelial elements (liver, bone marrow, lungs, and spleen) were identified as the major sites of catabolism of the transferrin variant. The liver took up more 59Fe from the variant (26% of the dose in 90 min) than from control rat transferrin (12%). The excess iron uptake was reduced by the intravenous injection of either human transferrin or ovalbumin, and it was abolished by administering both. Macrophages from bone marrow and lungs degraded the transferrin variant in vitro. The degradation was significantly enhanced when transferrin receptors were blocked by human transferrin, and it was significantly reduced by ovalbumin and methyl glucopyranoside.     

Levels and patterns of 125I-labeled transferrin binding in mouse embryonic teeth and kidneys at various developmental stages

The iron-transporting serum glycoprotein, transferrin, is necessary for the cell proliferation, morphogenesis, and differentiation of mouse embryonic teeth and kidneys in organ culture. The stimulatory effect of transferrin is mediated by the binding of transferrin to its specific cell-surface receptor and by receptor-mediated endocytosis. Since, in both teeth and kidneys, the requirement for and responsiveness to transferrin depend on the developmental stage of the organ, we studied the binding of transferrin at various stages of tooth and kidney development by incubating tissues with 125I-labeled transferrin. The amount of bound transferrin was determined by measuring the tissue-incorporated radioactivity, and the binding sites were localized by autoradiography. During tooth development in vitro, the requirement for exogenous transferrin is lost as the teeth proceed from the early cap stage to the bell stage. The level of transferrin binding was found to decrease simultaneously, and in bell-stage teeth, the transferrin receptors were concentrated in the areas of most active cell proliferation. In kidneys, the number of transferrin receptors was highest at the stage during which the undifferentiated kidney mesenchyme becomes responsive to transferrin. These receptors were located in both the ureter epithelium and the metanephric mesenchyme, and they dramatically decreased in number with advancing kidney differentiation. The results of the present study indicate that, during the embryonic development of teeth and kidneys, the amount and localization of transferrin binding are correlated with cell proliferation. The number of transferrin receptors is highest during the developmental stages when cell proliferation is most active, and decreases with advancing differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transplasmalemma electron transport from cells is part of a diferric transferrin reductase system

Intact cells are known to reduce external, impermeable electron acceptors. We now show that cells can reduce the iron in diferric transferrin at the cell surface and that this reduction reaction depends on the transferrin receptor as well as the transmembrane electron transport system. Reduction of external diferric transferrin is accompanied by oxidation of internal NADH which indicates that the transmembrane enzyme is an NADH diferric transferrin reductase. Highly purified liver plasma membranes have NADH diferric transferrin reductase activity which shows properties similar to the diferric transferrin reductases activity of intact cells. Cell growth stimulation by diferric transferrin and other impermeable oxidants which can react with the diferric transferrin reductase can be based on electron transport through he plasma membrane.     

Transferrin and iron in cultured chick embryonic neurons: a comparison between human and chick transferrins

Transferrin was not required for the short-term survival of cultured chick retinal neurons. Both human and chick transferrin failed to enhance the in vitro survival of 8- or 11-day embryonic chick retinal neurons when cultured in a defined medium. Furthermore, maintenance of neurons in the presence of chick transferrin antibody did not alter in vitro survival. Retinal neurons, however, could bind and internalize human or chick transferrin when assayed for by fluorescence immunohistochemical techniques. Binding and internalization of chick transferrin appeared to be greater than human transferrin. Iron uptake was measured in cultures maintained in the absence of transferrin. After incubation with 59FeCl3, iron uptake was 3.5 +/- 1.1 fmoles/cell. The presence of chick transferrin antibody did not significantly alter the amount of iron uptake occurring in this assay. In a comparison of human and chick transferrin mediated iron uptake, chick transferrin was 50% more effective than human transferrin in transporting iron. This study demonstrates that cultured embryonic retinal neurons are not dependent on transferrin for survival or iron uptake, although they actively bind and internalize transferrin. Results also demonstrate that whereas cultured chick retinal neurons can bind and utilize human transferrin, they do so with less efficiency than chick transferrin.     

Iron metabolism pathways in the rat hepatocyte

A small to moderate inhibitory effect of iron uptake by isolated rat hepatocytes in short-term studies was seen with oxidative phosphorylation and electron transport inhibitors, and no inhibition by agents affecting pinocytosis. Intracellular transferrin was able to donate iron to the small-molecular weight iron pool, and the latter was able to transfer, by a process not requiring energy or movement of serum transferrin, iron to ferritin. Serum transferrin was not able to lose iron to any cytosol components. Reducing agents were not able to abstract iron from rat serum transferrin to any great extent. It is concluded that iron is taken up by the rat hepatocyte from serum transferrin by a process not requiring energy or movement of serum transferrin into the cell interior; and that intracellular transferrin is involved in acquiring iron from serum transferrin at the cell surface, with iron then being transferred to the small-molecular weight iron pool and hence to ferritin. It is also proposed that intracellular transferrins may have the general function of interacting with serum transferrin at cell surfaces.     

Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells

Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.     

Transferrin uptake and release by reticulocytes treated with proteolytic enzymes and neuraminidase.

The mechanism of transferrin uptake by reticulocytes was investigated using rabbit transferrin labelled with 125I and 59Fe and rabbit reticulocytes which had been treated with trypsin, Pronase or neuraminidase. Low concentrations of the proteolytic enzymes produced a small increase in transferrin and iron uptake by the cells. However, higher concentrations or incubation of the cells with the enzymes for longer periods caused a marked fall in transferrin and iron uptake. This fall was associated with a reduction in the proportion of cellular transferrin which was bound to a cell membrane component solubilized with the non-ionic detergent, Teric 12A9. The effect of trypsin and Pronase on transferrin release from the cells was investigated in the absence and in the presence of N-ethylmaleimide which inhibits the normal process of transferrin release. It was found that only a small proportion of transferrin which had been taken up by reticulocytes at 37 degrees C but nearly all that taken up 4 degrees C was released when the cells were subsequently incubated with trypsin plus N-ethylmaleimide, despite the fact that about 80% of the 59Fe in the cells was released in both instances. Neuraminidase produced no change in transferrin and iron uptake by the cells. These experiments provide evidence that transferrin uptake by reticulocytes requires interaction with a receptor which is protein in nature and that following uptake at 37 degrees C, most of the transferrin is located at a site unavailable to the action of proteolytic enzymes. The results support the hypothesis that transferrin enters reticulocytes by endocytosis.     

Physical characterization of the transferrin receptor in human placentae

The physical properties and binding characteristics of the solubilized transferrin receptor isolated from the placental brush-border membrane of a human trophoblast cell were investigated. The receptor protein was isolated from solubilized 125I-labeled membranes by immunoprecipitation with anti-human transferrin in the presence of saturating amounts of human transferrin. Gel filtration on acrylamide agarose (AcA-22) at 23 degrees C in the absence of transferrin indicates the transferrin receptor has a Stokes radius of 4.6 nm. In the presence of transferrin, the Stokes radius of the receptor shifts to 6.3 nm. Sucrose density centrifugation studies indicate that it has a sedimentation coefficient of 9.8 S in the absence of transferrin and 11.2 S in the presence of transferrin. The molecular weight for the transferrin free receptor is calculated to be 213,000. Upon incubation with transferrin, it increases to 364,000. This is consistent with the idea that the active form of the solubilized receptor is a dimer and the dimer is in turn capable of binding two transferrin molecules.