Comparison of adriamycin and derivatives uptake into large unilamellar lipid vesicles in response to a membrane potential

The uptake of adriamycin (ADM) and several derivatives into large unilamellar vesicles (LUV) displaying a transmembrane potential and having a lipid composition close to that of the inner mitochondrial membrane has been measured. Drug association to neutral liposomes, made of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (70:30, w/w) was shown to be potential-dependent: in the absence of potential, accumulation of drug was almost undetectable, whereas between 11 and 50 nmol of drug/mumol phospholipid, depending on the anthracycline used, was associated to LUV exhibiting a membrane potential after 1 h incubation. Association of drugs to LUV with a lipid composition closer to that of the inner mitochondrial (cardiolipin, CL, 20%; PC 50%; PE, 30%, w/w) and displaying a membrane potential is higher than with neutral vesicles (between 40 and 76 nmol of anthracycline/mumol phospholipid after 1 h incubation). Since it is known that ADM and derivatives have a high affinity for CL, a fraction of the associated drug may bind to CL on the outer side of the vesicles. This was confirmed by the fact that, in the absence of potential, between 40 and 56 nmol of anthracycline/mumol phospholipid was still associated to LUV containing CL. In order to discriminate between drug adsorbed at the surface of the LUV and drug accumulated inside the LUV, an anthracycline fluorescence quencher (I-) was used. It was shown on neutral LUV displaying a membrane potential, that between 55 and 81% of the associated drug is actually entrapped inside the vesicles, inaccessible to the quencher. These percentages decreased to between 41 and 68%, respectively, in the presence of LUV containing CL and exhibiting a membrane potential, whereas for LUV of the same composition but displaying no membrane potential almost all the associated drug is adsorbed on the outer face of the LUV, accessible to the quencher, and likely bound to CL. This study brings evidence that antitumour anthracyclines despite important structural homologies do not accumulate to the same extent into vesicles mimicking the lipid composition and the membrane potential of mitoplasts. This ability to reach the matrix compartment of mitochondria could partly explain the differences of cardiotoxicities associated to anthracyclines with closely related molecular structure.     

The ability of verapamil to restore intracellular accumulation of anthracyclines in multidrug resistant cells depends on the kinetics of their uptake

The basic distinguishing feature of all cells expressing functional P-glycoprotein-multidrug resistance is a decrease of steady state drug levels as compared to those in drug-sensitive controls. A variety of small molecules, such as verapamil and cyclosporin A, bind to P-glycoprotein and inhibit its ability to pump out antitumor drugs. The kinetics of P-glycoprotein-mediated efflux of various anthracycline derivatives was measured in multidrug-resistant (MDR) K562 cells in the presence of verapamil. Used for the purpose were daunorubicin, idarubicin and 8-S-fluoro-idarubicin which have the same pKa of deprotonation equal to 8.4, but different lipophilicity, 4'-epi-2'-bromo-daunorubicin which has a lipophilicity which is comparable to that of daunorubicin but a pKa equal to 6.3, pirarubicin with pKa equal to 7.7 and lipophilicity different from that of these derivatives were used. Our data show (1) that verapamil is unable to completely block the P-glycoprotein-mediated efflux of anthracyclines and that 10% of its functionality remains even with high verapamil concentrations, (2) that the ability of verapamil to restore intracellular accumulation of anthracyclines in MDR cells depends on the kinetics of their uptake. With fast kinetics uptake, as is the case for idarubicin, 8-S-fluoro-idarubicin, 4'-epi-2'-bromo-daunorubicin and pirarubicin (which have either a low pKa and/or high lipophilicity), verapamil can restore in multidrug resistant cells an intracellular drug level which is comparable to that observed in sensitive cells. This is not possible when the kinetics of uptake is low as is the case for daunorubicin. Cyclosporin A is a more potent modulator and is able to fully restore daunorubicin accumulation in multidrug resistant cells.     

On the methods for studying the mechanisms and bioavailability of iron

Bullous pemphigoid (BP) blisters contain several molecules, some of which spread into the blisters from the interstitial fluid, while others are produced locally and migrate into the circulation. The calculation of the ratios between blister/serum concentrations may help to distinguish between these two types of molecules. The rules regulating the diffusion of the molecules have been described only in suction blisters, where the theoretical molecular weight (MW) represents one of the principal influencing factors. The aim of the present study was to analyse the relationship between theoretical MWs and the ratios of concentrations of several molecules evaluated both in sera and in blister fluids. Eight cytokines (interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-10, tumor necrosis factor-alpha, oncostatin-M and vascular endothelial growth factor), two acute phase reactants (alpha-1 acid glycoprotein, haptoglobin), albumin, one soluble membrane molecule with adhesion functions (sICAM-1) and the eosinophil cathionic protein (ECP) were measured in samples from 15 patients affected with BP by means of commercially available tests. The data suggest that the MW may influence the rate of diffusion throughout the blister, both in input and output directions, despite the discontinuity observed at the basement membrane level on the BP blister floor.     

The overall partitioning of anthracyclines into phosphatidyl-containing model membranes depends neither on the drug charge nor the presence of anionic phospholipids

Anthracyclines are potent anticancer agents. Their use is limited by the problem of multidrug resistance (MDR) associated with a decreased intracellular accumulation of drug correlated with the presence, in the membrane of resistant cells, of the P-glycoprotein responsible for an active efflux of the drug. The activity of a drug depends upon its intracellular concentration which itself depends on the kinetics (a) of passive influx (b) of passive efflux and (c) of the P-glycoprotein-mediated efflux of drug across the cell membrane. The ability of an anthracycline to overcome MDR depends largely on the first point. The passive drug uptake is governed by their incorporation into the lipid matrix and both electrostatic and hydrophobic forces seem necessary for the stabilization of anthracyclines into lipid bilayers. The aim of the present study was to determine the relative importance of these two interactions. Using microspectrofluorometry and the observation that the fluorescence of anthracycline is enhanced when the dihydroanthraquinone part is embedded within the lipid bilayer, we have determined the partition coefficient (alternatively, the binding constant) of 12 anthracycline derivatives in large unilamellar vesicles. The anthracyclines were (a) doxorubicin, daunorubicin and idarubicin which, at pH 7.2, bear a single positive charge at the level of the amino group on the sugar, (b) their corresponding neutral 3'-hydroxy derivatives where the amino group in the sugar has been replaced by a hydroxyl, (c) the three 13-hydroxy derivatives, doxorubicinol, daunorubicinol and idarubicinol, (d) pirarubicin and (e) two permanently positively charged derivatives. The large unilamellar vesicles contained phosphatidylcholine with various amounts of phosphatidic acid which is negatively charged and of cholesterol. We came to the conclusion that the efficiency of drug incorporation in the bilayers depends neither on the presence of a positive charge on the drug nor on the presence of anionic phospholipid but on the hydrophobicity of the molecule: the neutral and the positively charged form have the same ability to partition into the bilayer. However, the percentage of each form present should depend on the electrostatic parameters.     

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.     

Efficacy of synthetic and biological bioadhesives in scleral tunnel phacoemulsification in eyes with high myopia

The binding of several benzopiranone derivatives to human serum albumin was determined. The antiallergic drug disodium cromoglycate binds weakly to serum albumin. However, its precursors, chromones of smaller size, were able to bind in a hydrophobic pocket in the protein, and are carried by serum albumin in blood.     

Investigation of suramin-albumin binding by electrospray mass spectrometry

Suramin, an organic polyanion with six sulphonic groups, is under clinical trials as an agent against hormone-refractory prostate cancer. The drug binds strongly to serum albumin. The objectives were to use electrospray to measure the molecular masses of the intact complexes of albumin and suramin to determine the number of suramin molecules bound under different molar ratios, and to investigate the binding of suramin in human serum. With albumin in excess (2:1 to 25:1 ratio), only 1 and 2 bound suramins were found; with suramin excess (2:1 to 1000:1) up to 20 bound suramin molecules/albumin were found. Up to 5 bound suramins were found in human serum with 4:1 suramin:albumin ratio, which corresponds to recommended therapeutic doses (200-300 micrograms/mL). At 8:1 ratio, which would be toxic, complexes with up to ten bound suramin molecules were found, and unreacted albumin diminished.     

Intestinal water and solute absorption studies: comparison of in situ perfusion with chronic isolated loops in rats

The effects of lumenal glucose on jejunal water transport and the influence of glucose-induced water absorption on solute uptake from single-pass perfusions are compared in anesthetized rats in situ and isolated chronic loops in unanesthetized rats in vivo. While the magnitudes of solute membrane permeabilities are consistently higher in the chronic loop system, the effects on water transport and its promotion of jejunal solute uptake are comparable between the two experimental systems. The effect of glucose-induced water absorption on the enhanced/baseline jejunal uptake ratio of the hydrophilic drug, acetaminophen, is greater than that for the lipophilic drug, phenytoin, in both experimental systems. The fact that chronic loop effective solute permeabilities were equivalent to solute membrane permeabilities in situ is consistent with greater lumenal fluid mixing in vivo. In addition, in situ body temperature affects the uptake of phenytoin but not acetaminophen, water, or glucose. This suggests that active and paracellular solute transport is not compromised in situ, while membrane partitioning and diffusion of lipophilic species are more sensitive to experimental conditions.     

Kinetic analysis in living cells of the inhibition of the P-glycoprotein-mediated efflux of anthracyclines by vinca alkaloids

Cells that overexpress the mdr 1 gene have decreased steady-state accumulation and increased efflux of many anticancer drugs including anthracyclines and vinca alkaloids. The mechanism(s) of P-glycoprotein-mediated efflux of drugs is (are) still poorly understood. In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, the cellular accumulation has been examined of pirarubicin, doxorubicin and idarubicin alone and in conjunction with four vinca alkaloid derivatives--vinblastine, navelbine, vindesine and vincristine. The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds. Erythroleukemia K562 cell lines were used. It has been shown that the P-glycoprotein-mediated efflux of these three anthracyclines can be inhibited by vinca alkaloids derivatives. At pH 7.2, 50% of the P-glycoprotein-mediated efflux of daunorubicin and idarubicin was inhibited by about 40 +/- 10 microM vinblastine and that of pirarubicin by 10 +/- 2 microM vinblastine. The vinblastine concentration required to inhibit 50% of the active efflux of these anthracyclines did not depend on the anthracycline concentrations used, indicating that the inhibition was non competitive. The ability of navelbine, vincristine and vindesine to inhibit the active efflux of pirarubicin was also checked; 15 +/- 3 microM navelbine are required to inhibit 50% of the active efflux but at concentrations lower than 100 microM, neither vincristine nor vindesine were able to inhibit this efflux, indicating that the vinca alkaloids compounds which are the most efficient are the most lipophilic. For the four vinca alkaloids, the concentration required to inhibit 50% of the efflux was lower as the pH was higher. A detailed kinetics analysis of the P-glycoprotein-mediated efflux of pirarubicin in the presence of vinblastine indicates a non competitive inhibition with K(I) = 12 +/- 2 microM.     

Lymphotactin: a key regulator of lymphocyte trafficking during acute graft rejection

One strategy for improving the antitumor selectivity and toxicity profile of antitumor agents is to design drug carrier systems with suitable transport proteins. Thus, four maleimide derivatives of doxorubicin were bound to thiolated human serum albumin which differed in the stability of the chemical link between drug and spacer. In the maleimide derivatives, 3-maleimidobenzoic or 4-maleimidophenylacetic acid was bound to the 3'-amino position of doxorubicin through a benzoyl or phenylacetyl amide bond and 3-maleimidobenzoic acid hydrazide or 4-maleimidophenylacetic acid hydrazide was bound to the 13-keto position through a benzoyl hydrazone or phenylacetyl hydrazone bond. The acid-sensitive albumin conjugates prepared with the carboxylic hydrazone doxorubicin derivatives exhibited an inhibitory efficacy in the MDA-MB-468 breast cancer cell line and U937 leukemia cell line comparable with that of the free drug (using the BrdU-(5-bromo-2'-deoxyuridine)-incorporation assay and tritiated thymidine incorporation assay respectively, IC50 approximately 0.1-1 microM) whereas conjugates with the amide derivatives showed no or only marginal activity. These results demonstrate that antiproliferative activity depends on the nature of the chemical bond between doxorubicin and carrier protein. Acid-sensitive albumin conjugates are suitable candidates for further in vitro and in vivo assessment.     

Analysis of drug/plasma protein interactions by means of asymmetrical flow field-flow fractionation

PURPOSE: The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption. METHODS: Field-Flow Fractionation is defined as a group of pseudochromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy. RESULTS: Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), alpha 2-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothiocyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA. CONCLUSIONS: If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug/plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation time.     

Uptake of basic drugs into rat lung granule fraction in vitro

Although basic drugs are distributed widely in various tissues, they are characteristically concentrated in the lung granule fraction. We examined the uptake of seven lipophilic basic drugs into rat lung granule fraction (P2) in vitro and investigated the contributions of drug lipophilicity and lysosomal trapping to the characteristic lung P2 distribution. The uptake of each drug into P2 was examined at various pH values. The drug concentration in P2 was determined by gas chromatography. Biperiden (BP) was rapidly taken up into P2, reaching a maximal concentration within 1 min at pH 7.4 at both 4 degrees C and 37 degrees C. Both BP and chlorpromazine uptake into P2 was biphasic. Though the uptake rates of the seven drugs into P2 increased with rising pH, the rate of increase varied for each drug. There was a good correlation between the octanol-water partition coefficient of the non-ionized form (P(oct)) of each drug and the uptake into P2 in the presence or absence of NH4Cl, which inhibits lysosomal trapping. However, uptake into P2 in the presence of NH4Cl showed a stronger P(oct)-dependency. We conclude that the distribution of basic drugs into lung P2 is dependent on both drug lipophilicity and lysosomal uptake.     

Interaction of phosphatidylcholine with bovine serum albumin. Specificity and properties of the complexes

Phosphatidylcholine dispersed on Celite was rapidly solubilized by neutral bovine serum albumin solutions. Stable protein-lipid complexes were isolated by Agrose gel filtration or by ultracentrifugal flotation in high density solvents, and the physicochemical properties of the complexes were investigated in terms of the stoichiometry of binding, effect of fatty acid ligands on phosphatidylcholine binding, effect of high ionic strength on the stability of the complexes, intrinsic fluorescence and circular dichroism spectra, and sedimentation velocity coefficients. Complexes containing from 2 to 30 phosphatidylcholine molecules per protein molecule were observed; however, no saturation of binding sites could be detected in this range of molar ratios. Oleic acid binding by serum albumin prevents interaction of the protein with phosphatidylcholine, indicating possible competition of these ligands at low contents of the phospholipid. For molar ratios of up to 10 phosphatidylcholine molecules per serum albumin, binding is primarily due to hydrophobic interactions that have no effect on the overall shape and secondary structure of the native protein except for local modifications at tryptophan residues, whose fluorescence becomes quenched and blue shifted on phosphatidylcholine binding. Similar phosphatidylcholine uptake experiments performed with a series of globular proteins indicated that the lipid extraction from Celite surfaces is a non-specific process, accelerated by several other proteins (e.g. aldolase, egg albumin, chymotrypsinogen, soybean trypsin inhibitor, and the major apolipoprotein from bovine serum high density lipoprotein). Formation of stable protein-lipid complexes, however, was only observed with bovine serum albumin, which in contrast to the other proteins is known to have affinity binding sites for anions with hydrophobic side chains.     

Albumin's role in steroid hormone action and the origins of vertebrates: is albumin an essential protein?

Albumin, the major serum protein, binds a wide variety of lipophilic compounds including steroids, other lipophilic hormones and phytochemicals that bind to hormone receptors. Albumin has a low affinity for these lipophilic compounds. However, due to albumin's high concentration in serum, albumin is a major carrier of steroids and lipophilic hormones and regulator of their access to their receptors. Moreover, albumin functions as a sink for phytochemicals, which prevents their binding to hormone receptors and other cellular proteins, protecting animals from disruptive phytochemical-mediated endocrine effects. We propose that these properties of albumin were important in protochordates and vertebrates about 550 to 520 million years ago, just before and during the Cambrian. At that time, animal body sizes and exposure to phytochemicals in food were increasing, and animals in which albumin expression was high had a selective advantage in surviving and reproducing in the presence of toxic phytochemicals. This hypothesis that albumin has essential function(s) in mammalian endocrine physiology can be tested by comparing the effects of phytochemicals in Nagase rats that have 1/1000 the normal albumin concentration or in mice in which the albumin gene is knocked out with those in normal rats and mice.     

Pharmacokinetics of methotrexate-albumin conjugates in tumor-bearing rats

Linking chemotherapeutic drugs to a macromolecular carrier system may enhance tumor targeting, reduce toxicity and overcome drug resistance mechanisms. As an elementary model to evaluate the pharmacological properties of macromolecular drug carrier systems we chose rat serum albumin (RSA) for carrier and methotrexate (MTX) as antineoplastic drug. The conjugation procedure yielded conjugates with an approximate 1:1 molar loading rate (MTX(1)-RSA). In the first part of the study a residualizing [111In]DTPA protein label was used for mapping in vivo the catabolic sites of the native carrier protein and of the MTX(1)-RSA drug conjugate in Walker 256 carcinosarcoma bearing rats. The tumor accumulation was about 14% of the injected dose for the RSA and MTX(1)-RSA tracers after 24 h. Tracer entrapment by organs with an active mononuclear phagocyte system was low (liver below 7% and spleen below 1.5% of the injected dose after 24 h). The 1:1 conjugation of MTX to RSA did not decisively alter the pharmacokinetic properties nor the tumor or tissue distribution of the native carrier protein RSA. In the second part of the study the different properties of the MTX(1)-RSA conjugate were compared with MTX in vivo. About 2 mg MTX/kg body weight either of the drug conjugate or of the original drug were injected after being additionally spiked with radiolabeled tracers. Plasma concentrations were simultaneously determined by immunological and radioactive means. After 24 h about 12% MTX(1)-RSA was found in circulation compared to 0.03% MTX. Favorable tumor accumulation rates of about 14% were achieved for MTX(1)-RSA versus 0.04% for MTX. About 45-fold more of the injected dose of [3H]MTX accumulated in the liver as compared to the tumor (1.5 versus 0.03%) after 24 h. Conjugation of MTX to RSA reversed this ratio in favor of the tumor to 1:1.4 (13.6 versus 9.6%). In conclusion, the potential therapeutic benefit of the MTX(1)-RSA conjugate lies in its very long tumor exposure time and its improved tumor accumulation rate compared to conventional MTX. In addition the conjugation to albumin might enhance the therapeutic effects over those achieved by long-term continuous infusion of MTX, as MTX(1)-RSA enters the cells by a different uptake mechanism. This might also help to circumvent MTX resistance mechanisms, such as a reduction in folate receptor numbers or impaired MTX polyglutamylation.     

Kinetics of the acute-phase reaction in rats after tumor transplantation

Transplantation of Yoshida sarcoma (solid type) and Zajdela ascites hepatoma tumors in rats induces a biphasic change in the concentration of the following five acute-phase proteins: alpha-1-acid glycoprotein; alpha-1-antitrypsin; haptoglobin; hemopexin; and ceruloplasmin. These proteins and other plasma proteins were quantitated by two-dimensional immunoelectrophoresis relative to normal serum concentrations. The elevation of most of these acute-phase proteins was greater in the second phase, during which serum levels increased continuously as the tumor burden increased until the animals died. The increase in haptoglobin concentration during the second phase was much higher in rats bearing Yoshida sarcoma than in rats bearing Zajdela tumors. Rats receiving irradiated tumor cells showed neither tumor growth nor second-phase protein changes. Significant increases in uptake of 3H-amino acids by isolated perfused livers of tumor-bearing rats provided evidence for an increase in the hepatic synthesis rates of the acute-phase proteins. Removal of the solid tumor resulted in a gradual decrease of acute-phase protein concentrations with concomitant increase in serum albumin concentration. These alterations in serum acute-phase proteins during tumor growth and after removal of the tumor may make their use attractive as biological markers of the response of the tumor-bearing animal to its tumor.     

Effect of charged groups on the adsorption and penetration of proteins onto and into carboxymethylated poly(HEMA) hydrogels

A range of carboxymethylated poly(hydroxyethyl methacrylate) (CM-PHEMA) hydrogels with varying degrees of carboxymethylation was synthesized for a systematic study of the effects of ionized groups ('charge') on the uptake by hydrogel matrices of the proteins, lysozyme and human serum albumin (HSA). Using a radiolabel-tracer technique, X-ray photoelectron spectroscopy, and laser scanning confocal microscopy, we attempted to differentiate between protein molecules that were irreversibly adsorbed onto the hydrogel surface and those that penetrated into the hydrogel matrix. The effective pore size of the CM-PHEMA hydrogels was modelled and compared with the known molecular dimensions of the two proteins. The effects of the presence of varying amounts of ionized groups in the hydrogel matrix differed for the two proteins. For lysozyme, increased uptake was observed at higher carboxymethylation; this is interpreted as resulting from a combination of electrostatic attraction and increasing ease of penetration of the protein into the more porous hydrogel matrix. For HSA, on the other hand, the uptake was primarily by surface adsorption, with little diffusive penetration into the matrix.     

Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione

The M(r) 190,000 multidrug resistance protein (MRP) confers resistance to a broad spectrum of natural product drugs. However, it has not been possible to demonstrate that MRP can actively transport unmodified forms of these compounds, although the protein has been shown to transport structurally diverse glutathione (GSH)- and glucuronide-conjugated molecules. Previously, we showed that ATP-dependent uptake of vincristine by MRP-enriched, inside-out membrane vesicles could be stimulated by physiological concentrations of GSH (Loe et al., J. Biol. Chem., 271: 9675-9682, 1996). We have now established that the ATP/GSH dependent vincristine uptake is both proportional to the level of MRP in the membrane vesicles and can be inhibited by monoclonal antibodies shown previously to inhibit transport of established MRP substrates, such as leukotriene C4. We also show that short-chain alkyl derivatives of GSH can stimulate drug uptake, which suggests that vincristine transport does not necessarily involve a change in redox state or glutathionylation of the protein. Furthermore, vincristine uptake is accompanied by ATP- and drug-dependent accumulation of GSH, which can also be stimulated to a lesser extent by vinblastine but not daunorubicin or doxorubicin. Although GSH or vincristine alone are very poor inhibitors of MRP-mediated transport of leukotriene C4, together they act as relatively potent competitive inhibitors. Overall, our data demonstrate that MRP can actively cotransport GSH and unmodified vincristine and that these compounds probably interact, either with the leukotriene C4 binding site(s) on the protein or with a mutually exclusive site.     

Chemical modification and photograft polymerization upon expanded poly(tetrafluoroethylene)

The objective of this investigation was to characterize intranuclear accumulation of oligonucleotides and their adducts with non-karyophilic compounds in cultured animal cells and thus to present a model system for nucleic acid-mediated nuclear import. In digitonin-permeabilized cells, nuclear uptake of 3'-FITC-labeled, single-stranded 25-mer oligodeoxyribonucleotides was independent of added cytosolic protein, largely energy-dependent, inhibitable by wheat germ agglutinin but not by N-ethylmaleimide, and a function of their base composition. When coupled to FITC-labeled streptavidin or streptavidin-bovine serum albumin conjugates, the oligonucleotides delivered the proteins to the nuclear interior with rates roughly proportional to their karyophilicity as free molecules. Transport activity was also demonstrated for single-stranded oligoribonucleotides. The transport was energy-dependent, inhibited by GMP-PNP and wheat germ agglutinin, but unaffected by N-ethylmaleimide. Nuclear import of oligo(dG)25/protein adducts needed 3 to 4 oligonucleotide signals per complex and the signal had to be at least 15 nucleotides long. Micro-injection experiments showed that the results obtained with digitonin-permeabilized cells are not artifacts of a quasi-intact cellular system. These data were confirmed by electron microscopy employing complexes of oligodeoxyribonucleotides with streptavidin-peroxidase-bovine serum albumin-1 nm gold. In permeabilized cells, the complexes docked to the cytoplasmic face of the nuclear pore complexes, were translocated through the central pore channel and accumulated in large quantities in the nuclear baskets before they were released into the nucleoplasm. These results demonstrate that nuclear uptake of oligonucleotides and their complexes is an active process mediated by nuclear pore complexes, which, at least regarding its cytoplasmic component, is different from the pathway requiring classical nuclear localization signals.     

Etiology and therapy of multiple organ failure

Uptake of persistent lipophilic toxicants in fish occurs via the food and by transfer across the body surface, notably the gills. Flux rates of most lipid soluble toxicants across the gills is rapid and the animal must eat at very high rates for feeding to have a significant effect on toxicant concentration in the body. The relative rates of uptake via feeding and transfer across the gills are analyzed from a theoretical and experimental standpoint. At the low feeding rates typical of fish, the uptake of toxicants in the food can be ignored when estimating toxicant body concentration.