Ioav Cabantchik | The Hebrew University of Jerusalem (original) (raw)

Papers by Ioav Cabantchik

Proceedings of The National Academy of Sciences, 1985

Chloroquine (CQ) accumulates in the acidic food vacuole of intraerythrocytic malaria parasites (P... more Chloroquine (CQ) accumulates in the acidic food vacuole of intraerythrocytic malaria parasites (Plasmodium falciparum) by virtue of its weak base properties. In the present work, the extent of CQ accumulation was determined by the transvacuolar pH gradient: modification of the lattereither by changing the external pH or by adding the acidotropic agent NH4Cl-led to a corresponding change in CQ distribution between cells and medium. Changes in pH gradient provoked a change in the susceptibility of parasites to CQ: at external pH values of 8.0, 7.4, and 6.8, the IC50 values for CQ were 0.48 x 10-7 M, 1.8 x 10-7 M, and 3.3 x 10-7 M, respectively. Marked resistance to CQ (IC50 = 9.8 x 10-7 M) was conferred upon cells by exposing them simultaneously to CQ and 10 mM NH4Cl, at pH 7.4. The final concentration of CQ

Febs Letters, 1997

Cell iron status was assessed in terms of its capacity to mediate cell injury by pro-oxidants. Cu... more Cell iron status was assessed in terms of its capacity to mediate cell injury by pro-oxidants. Cultured K562 cells, which maintain a stable cytosolic labile iron pool (LIP) of < 0.5 |xM, underwent distinct changes after short exposures to transferrin (Tf) followed by f-butyl hydroperoxide (TBHP) : (a) rise in LIP, detectable fluorimetrically; (b) increased lipid peroxidation and (c) eventual cell death. All of these effects were inhibited by weak bases or iron chelators. Similarly, hydrogen peroxide caused rises in both LIP and oxidant species detectable with 2',7'-dichlorofluorescin diacetate, which were enhanced by preincubation with Tf. The Tf-delivered iron disappeared from LIP and the TBHP-reactive pool with a t| /2 < 30 min. The results indicate that the catalytic potential of iron is highest while in transit between endosomes and cytosolic ligands.

Journal of Laboratory and Clinical Medicine, 2001

Although iron chelation therapy results in a significant improvement in well-being and life expec... more Although iron chelation therapy results in a significant improvement in well-being and life expectancy of thalassemic patients with transfusional iron overload, failure to achieve these goals in a substantial proportion of patients underlines the need for improved methods of treatment. In the present studies we used selective radioactive iron probes of hepatocellular and reticuloendothelial (RE) iron stores in hypertransfused rats and iron-loaded heart cells to compare the source of iron chelated in vivo by deferoxamine (DFO) or by deferiprone (L1) and its mode of excretion, to examine the ability of DFO and L1 to remove iron directly from ironloaded myocardial cells, and to examine the mechanism of their combined interaction through a possible additive or synergistic effect. Our results indicate that L1 given orally is 1.6 to 1.9 times more effective in rats, on a weight-per-weight basis, than parenteral DFO in promoting the excretion of storage iron from parenchymal iron stores but shows no advantage over DFO in promoting RE iron excretion. Simultaneous administration of DFO and L1 results in an increase in chelating effect that is additive but not synergistic. The magnitude of this additive effect is identical to an increase in the equivalent weight or molar) dose of DFO alone rather than the sum of the separate effects of L1 and DFO. This finding is most probably the result of a transfer of chelated iron from L1 to DFO. These observations may have practical implications for current efforts to design better therapeutic strategies for the management of transfusional iron overload. (J Lab Clin Med 2001;138:130-8) Abbreviations: DFO = deferoxamine; DRBC = heat-damaged red blood cell; Fl-DFO = fluorescein-DFO; HBS = HEPES-buffered-saline solution (150 mmol/L NaCl, 20 mmol/L N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.3); L1 = deferiprone; RE = reticuloendothelial

Febs Letters, 1996

The labile iron pool of cells (LIP) constitutes the primary source of metabolic and catalytically... more The labile iron pool of cells (LIP) constitutes the primary source of metabolic and catalytically reactive iron in the eytosol. We studied LIP homeostasis in K562 cells using the fluorescent metal-sensitive probe calcein. Following brief exposure to iron(ll) salts or to oxidative or reductive stress, LIP rose by up to 120% relative to the normal level of 350 nM. However, the rate of recovery to normal LIP level differed markedly with each treatment (respective tl/2s of 27, 65-88 and -<17 min). We show that the capacity of K562 cells to adjust LIP levels is highly dependent on the origin of the LIP increase and on the pre-existing cellular iron status.

Analytical Biochemistry, 1996

Iron chelators are important tools in biochemical studies of iron metabolism and in the therapy o... more Iron chelators are important tools in biochemical studies of iron metabolism and in the therapy of iron overload diseases. Their mode of action is comprised of entry into cells and scavenging intracellular metal, which includes complexation and egress of the complex. Iron is a metal which appears in the cells in various chemical forms and in different compartments. The form of the metal directly affected by the chelators is the most labile, low-molecular-weight type, which is present in the cytosol and is known as the "chelatable iron." This form is thought to be in dynamic equilibrium with several sequestered forms present in the cell, including the iron-responsive proteins. We recently introduced a fluorescent method for assessing the chelatable iron pool of cells, based on the quenching of the fluorescent calcein by metal ions (Breuer et al., J. Biol. Chem., 1995, 270, 24209-24215). In this work we adapted the method for dynamic assessment of chelator efficacy in scavenging iron from cells. In assay 1, red blood cells ghosts are used as a cell membrane model. The free-acid (impermeant) form of calcein is loaded into ghosts by encapsulation (lysis and resealing) and its fluorescence is quenched by addition of permeant iron(II). Chelators added to ghosts lead to iron removal from calcein and hence to recovery of fluorescence, commensurate with their permeation into ghosts and iron binding affinity. In assay 2, human K562 erythroleukemia cells are loaded with calcein via its permeating and cleavable acetoxymethyl form. A fraction of the intracellular calcein fluorescence is quenched in situ by endogenous cellular iron. The rate of dequenching which is obtained after addition of a chelator provides a measure for the scavenging of the intracellular metal, a process which, as in assay 1, depends on chelator permeation and binding affinity for iron. The two methods provide convenient means for assessing the efficacy of candidate chelator structures in depleting cell iron pools. They are also potentially applicable to chelation of other metals such as Co(II), Ni(II), and Cu(II) and to any cellular system or membrane vesicles.

Journal of Membrane Biology, 1972

The disulfonic acid stilbene derivative SITS reported to be covalently bonded to the membrane of ... more The disulfonic acid stilbene derivative SITS reported to be covalently bonded to the membrane of the red blood cell, was found to be largely reversibly bound. Reversal of its specific inhibitory effect on anion permeability was attained by washing the cells with buffer containing albumin. The small fraction of covalently bonded SITS could be increased by prolonging the time of exposure of the cells or by multiple exposures. A series of other disulfonic stilbene derivatives was synthesized. All of them specifically inhibited anion permeability whether or not they are capable of forming covalent bonds. Their inhibitory effectiveness, however, varied over a 5,000-fold range, allowing certain conclusions to be made concerning the chemical architecture of the binding site. Certain of the compounds were almost entirely covalently bonded. One of them was labeled with125I and used to determine to which membrane proteins the compound is bound. Over 90% was found in a protein band on acrylamide gels of 95,000 mol wt. The most effective compound against sulfate permeability was equally effective against chloride permeability, producing a maximum inhibition of over 95%. The residual anion fluxes respond differently to pH and temperature than do the fluxes of unmodified cells.

Analytical Biochemistry, 2005

Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Vari... more Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Various methods to measure NTBI were comparatively assessed as part of an international interlaboratory study. Six laboratories participated in the study, using methods based on iron mobilization and detection with iron chelators or on reactivity with bleomycin. Serum samples of 12 patients with hereditary (n D 11) and secondary (n D 1) hemochromatosis were measured during a 3-day analysis using 4 determinations per sample per day, making a total of 144 measurements per laboratory. Bland-Altman plots for repeated measurements are presented. The methods diVered widely in mean serum NTBI level (range 0.12-4.32 mol/L), between-sample variation (SD range 0.20-2.13 mol/L and CV range 49.3-391.3%), and within-sample variation (SD range 0.02-0.45 mol/L and CV range 4.4-193.2%). The results obtained with methods based on chelators correlated signiWcantly (R 2 range 0.86-0.99). On the other hand, NTBI values obtained by the various methods related diVerently from those of serum transferrin saturation (TS) when expressed in terms of both regression coeYcients and NTBI levels at TS of 50%. Recent studies underscore the clinical relevance of NTBI in the management of iron-overloaded patients. However, before measurement of NTBI can be introduced into clinical practice, there is a need for more reproducible protocols as well as information on which method best represents the pathophysiological phenomenon and is most pertinent for diagnostic and therapeutic purposes. 

Transfusion Science, 2000

The concept of non-transferrin bound iron (NTBI) was introduced 22 years ago by Hershko et al. (B... more The concept of non-transferrin bound iron (NTBI) was introduced 22 years ago by Hershko et al. (Brit. J. Haematol. 40 (1978) 255). It stemmed from a suspicion that, in iron overloaded patients, the large amounts of excess iron released into the circulation are likely to exceed the serum transferrin (Tf) iron-binding capacity (TIBC), leading to the appearance of various forms of iron not bound to Tf. In accordance with this assumption, NTBI was initially looked for and detected in patients with > or = 100% Tf-saturation. As techniques for its detection became more sophisticated and sensitive, NTBI was also found in conditions where Tf was not fully saturated, leading to a revision of the original view of NTBI as a simple spillover phenomenon. In this review, we will discuss some of the properties of NTBI, methods for its detection, its significance and potential value as an indicator for therapeutic regimens of iron chelation and supplementation.

Analytical Biochemistry, 2001

We introduce a method for monitoring non-transferrin-bound iron (NTBI), a labile and potentially ... more We introduce a method for monitoring non-transferrin-bound iron (NTBI), a labile and potentially toxic form of serum iron associated with imbalanced iron metabolism. The assay employs fluorescein-labeled apotransferrin (Fl-aTf), which undergoes fluorescence quenching upon binding iron. It has the advantages of simplicity, high sensitivity, and detection of those forms of NTBI that persist in sera with low transferrin saturations. Since NTBI is not readily available for detection, it is mobilized by 10 mM oxalate. Endogenous serum apotransferrin, capable of binding oxalate-mobilized NTBI, is blocked by 0.1 mM gallium(III). This metal, like iron, binds to Fl-aTf, but it neither quenches its fluorescence nor interferes with quenching by iron. Serum and reagent containing oxalate, Ga(Cl) 3 , and Fl-aTf are mixed in multiwell plates and fluorescence is determined after 1 h in a microplate reader. To compensate for artifactual fluorescence changes caused by serum color, parallel samples are prepared with excess unlabeled apotransferrin, which scavenges all iron in the sample. Sera from eight hemochromatosis patients were tested for NTBI by the present assay and by an established alternative method, with qualitatively similar results. A potential application of the test is for screening large numbers of samples from patients at risk of developing NTBI.

European Journal of Clinical Investigation, 2002

Labile plasma iron (LPI) associated with iron supplementation has been implicated in complication... more Labile plasma iron (LPI) associated with iron supplementation has been implicated in complications found in dialysis patients. As LPI can potentially catalyse oxygen radical generation, we determined the presence of labile iron in the parenteral preparations and the frequency of occurrence of LPI in dialysis patients. The capacity to donate iron to apotransferrin (apo-) or to the chelator desferrioxamine (DFO) was measured with fluorescein-Tf (Fl-Tf) and Fl-DFO, respectively. Those probes undergo quenching upon binding to iron. Iron-catalysed generation of oxidant species was determined with dihydrorhodamine. Plasma nontransferrin-bound iron (NTBI), here termed LPI, was determined by mobilization of iron from low-affinity binding sites with oxalate, followed by its quantification with Fl-Tf in the presence of Ga(III). Normal individuals and most (80%) dialysis patients, analysed at least 1 week after iron supplementation showed no detectable (<0.2 microm) LPI. However, approximately 20% of the patients (n = 71) showed significant LPI levels (>0.2 microm), in some cases weeks after iron administration. LPI levels correlated best (r2 = 0.9) with Tf saturation. The iron preparations contained 2-6% low molecular weight and redox-active iron, most of which is chelated by Tf. Parenteral iron formulations contain a small but significant fraction of redox-active iron, most of which is scavenged by apo-Tf within <1 h. Therefore, oxidant stress associated with iron infusion is likely to be transient. The bulk of the polymeric iron is apparently inaccessible to apo-Tf. Although LPI might return to normal within 2 h of intravenous iron infusion, the long-term persistence of low-level LPI in up to 20% of end stage renal disease (ESRD) patients indicates that complete clearance of the intravenous iron may be more protracted than originally estimated.

Journal of Clinical Investigation, 1993

We designed the N-methylanthranilic-desferrioxamine (MA-DFO) as a fluorescent iron(III) chelator ... more We designed the N-methylanthranilic-desferrioxamine (MA-DFO) as a fluorescent iron(III) chelator with improved membrane permeation properties. Upon binding of iron(III), MA-DFO fluorescence is quenched, thus allowing traceability of drug-iron(III) interactions. MA-DFO is well tolerated by mammalian cells in culture. Its antimalarial activity is pronounced: IC50 values on in vitro (24-h) growth of Plasmodium fakciparum were 3±1 MM for MA-DFO compared with 30±8 for DFO. The onset of growth inhibition of rings or trophozoites occurs 2-4 h after exposure to 13 gM MA-DFO. This effect is commensurate with MA-DFO permeation into infected cells. In a 24-h exposure to MA-DFO or DFO, trophozoites take up either compound to -10% of the external concentration, rings to 5%, and noninfected cells to < 1%. Red cells encapsulated with millimolar concentrations of DFO or MA-DFO fully support parasite invasion and growth. We conclude that extracellular MA-DFO and DFO gain selective access into parasites by bypassing the host. The rate-limiting step is permeation through the parasite membrane, which MA-DVO accomplishes faster than DFO, in accordance with its iigher hydrophobicity. These views are consistent with the proposed duct, which apparently provides parasitized cells with a window to the external medium. (J. Clin. Invest. 1993. 91:218-224.)

Molecular Membrane Biology, 1979

The anion exchange system of human red blood cells is highly inhibited and specifically labeled b... more The anion exchange system of human red blood cells is highly inhibited and specifically labeled by isothiocyano derivatives of benzene sulfonate (BS) or stilbene disulfonate (DS). To learn about the site of action of these irreversibly binding probes we studied the mechanism of inhibition of anion exchange by the reversibly binding analogs p-nitrobenzene sulfonic acid (pNBS) and 4,4'-dinitrostilbene-disulfonic acid (DNDS). In the absence of inhibitor, the self-exchange flux of sulfate (pH 7.4, 25 degrees C) at high substrate concentration displayed self-inhibitory properties, indicating the existence of two anion binding sites: one a high-affinity transport site and the other a low-affinity modifier site whose occupancy by anions results in a noncompetitive inhibition of transport. The maximal sulfate exchange flux per unit area was JA = (0.69 +/- 0.11) X 10(-10) moles . min-1 . cm-2 and the Michaelis-Menten constants were for the transport site KS = 41 +/- 14 mM and for the modifier site Ks' = 653 +/- 242 mM. The addition to cells of either pNBS at millimolar concentrations or DNDS at micromolar concentrations led to reversible inhibition of sulfate exchange (pH 7.4, 25 degrees C). The relationship between inhibitor concentration and fractional inhibition was linear over the full range of pNBS or DNDS concentrations (Hill coefficient n approximately equal to 1), indicating a single site of inhibition for the two probes. The kinetics of sulfate exchange in the presence of either inhibitor was compatible with that of competitive inhibition. Using various analytical techniques it was possible to determine that the sulfate transport site was the target for the action of the inhibitors. The inhibitory constants (Ki) for the transport sites were 0.45 +/- 0.10 microM for DNDS and 0.21 +/- 0.07 mM for pNBS. From the similarities between reversibly and irreversibly binding BS and DS inhibitors in structures, chemical properties, modus operandi, stoichiometry of interaction with inhibitory sites, and relative inhibitory potencies, we concluded that the anion transport sites are also the sites of inhibition and of labeling of covalent binding analogs of BS and DS.

Biochimica Et Biophysica Acta-biomembranes, 1982

During the intraerythrocytic growth of Plasmodium falciparmn in culture, marked changes are obser... more During the intraerythrocytic growth of Plasmodium falciparmn in culture, marked changes are observed in the permeability properties of the host cell membrane. Anionic substances otherwise impermeant to normal cells, become highly permeant to infected cells. These changes in permeability become apparent as rings mature into trophozoites and remain throughout schizogony. The permeability changes to anionic substances are not manifested as degradation of band 3, the purported erythrocyte anion transporter. They probably reflect alterations of a more general nature.

Journal of Cellular Physiology, 1977

The nucleoside transport of systems of hamster cells are susceptible to inhibition by S-6-substit... more The nucleoside transport of systems of hamster cells are susceptible to inhibition by S-6-substituted derivatives of mercaptonucleosides. The mechanism of interaction between the most potent inhibitor of the class, 6-nitrobenzyl mercaptoinosine (NBMI) and the uridine transport system of hamster fibroblasts is studied in the present work. A kinetic description of the interaction is presented. Uridine transport is inhibited in a partially competitive fashion, leaving a substantial free fraction of the transport (20–30%) virtually insensitive to increasing concentrations of inhibitor. One interpretation compatible with the kinetic and chemical properties of the system assumes that binding of the inhibitor to carriers occurs at sites different from the substrate binding sites (allosteric binding). Such a binding induces a conformational change in the carrier as manifested in a reduced affinity to the substrate and a susceptibility to inhibition by organomercurials. The alternative interpretation postulates two parallel transport systems which display distinctly different susceptibilities to NBMI and to organomercurials. Experiments performed with non-penetrating organomercurials show that the sulfhydryl groups related either to the alleged allosteric components or to additional carriers, are located superficially on the membrane. The binding of NBMI is reversible, the affinity is extremely high (Ki = 0.15 nMolar) and the rate of reaction could probably be diffusionally limited at low concentrations of reagent (activation energy 250 cal/mole, rate k = 1.3.108 min−1. Molar−1). The high affinity properties of the probes are used to determine the number of NBMI binding sites. A value of 47,000 and 77,000 sites/cell was obtained by two separate methods.The possibility that allosteric properties are present in carrier systems are discussed in terms of current concepts of modulation of transport functions in biological membranes.

Planta, 1993

We demonstrate in this work that HCO inf3sup−uptake in the marine macroalga Ulva sp. features fun... more We demonstrate in this work that HCO inf3sup−uptake in the marine macroalga Ulva sp. features functional resemblances to anion transport mediated by anion exchangers of mammalian cell membranes. The evidence is based on (i) competitive inhibition of photosynthesis by the classical red-blood-cell anion-exchange blockers 4,4′-dinitrostilbene-2,2′-disulfonate and 4-nitro-4′-isothiocyanostilbene-2,2′-disulfonate under conditions where HCO inf3sup−, but not CO2, was the inorganic carbon form taken up; (ii) inhibition of HCO inf3−uptake by pyridoxal phospate, indicating the involvement of lysine residues in the binding/translocation of HCO inf3sup−; and (iii) inhibition of HCO inf3sup−(but not of CO2) uptake by exofacial trypsin treatments, indicating the functional involvement of a plasmalemma protein. It is suggested that HCO inf3sup−uptake mediated by such a putative anion transporter can be a fundamental step in providing inorganic carbon for the CO2-concentrating system of marine marcoalgae in an environment where the HCO inf3sup−concentration is high, but the CO2 concentration and rates of uncatalyzed HCO inf3sup−dehydration are low.

Journal of Cellular Physiology, 1985

Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in... more Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parastie growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appers as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.