Structural and magnetic properties of some lanthanide (Ln = Eu(iii), Gd(iii) and Nd(iii)) cyanoacetate polymers: field-induced slow magnetic relaxation in the Gd and Nd substitutions.

The lanthanide(iii) cyanoacetate complexes of the formula {[Ln2(CNCH2COO)6(H2O)4]·2H2O}n, where Ln = Eu (), Gd (), Nd (), have been prepared and characterized by X-ray diffraction analysis. Complexes and are isostructural and differ from the binding scheme of the neodymium compound , structurally described earlier. In all cases, the cyano group of the cyanoacetate ligand is not coordinated to the lanthanide cation. The carboxylic groups exhibit different binding modes: 2-bidentate-chelating, 2-bidentate and 2-tridentate-chelating bridging for and , and 4-bidentate and 2-tridentate-chelating bridging for the complex . The Eu compound shows field induced paramagnetism, as expected for a non-magnetic ground state with mixing from higher states. Combining the dc magnetization and luminescence measurements the spin-orbit coupling constant λ = 343 ± 4 cm(-1) was found, averaged over the two different sites for Eu in the lattice. In the Gd complex , a crystal field splitting of D/kB = -0.11 ± 0.01 K has been found for the S = 7/2 multiplet of the Gd(iii) ion. No slow relaxation at H = 0 is observed because the low anisotropy barrier allows fast spin reversal through classical processes. The application of an external magnetic field induces two slow relaxation processes. It is argued that the first relaxation rate is caused by the resonant phonon trapping (RPT) mechanism, while the second, slower relaxation rate is due to the lifting of the Kramers degeneracy on the ground state. For compound heat capacity and dc susceptibility measurements indicate that at very low temperatures the ground state Kramers doublet has strong single ion anisotropy. The energy to the next excited doublet ΔZFS/kB = 104 K has been calculated by ab initio calculation methods. The g* tensor has also been calculated, showing that it has predominant anisotropy along the z-axis, and there is an important transversal component. At H = 0 quantum tunnelling is an effective mechanism in producing a fast relaxation to equilibrium at temperatures above 1.8 K. The dipolar or exchange interactions and a sizable transverse anisotropy component in the ground state enhance the quantum tunneling probability. Under an external applied field, two slow relaxation processes appear: above 3 K the first relaxation mechanism is of the Orbach type, with an activation energy U/kB = 27 K; the slower relaxation is caused by the direct relaxation process from the ground state to the Kramers split levels by the applied field.

GABAA receptor associated protein (GABARAP) modulates TRPV1 expression and channel function and desensitization.

Transient receptor potential vanilloid (TRPV1) transduces noxious chemical and physical stimuli in high-threshold nociceptors. The pivotal role of TRPV1 in the physiopathology of pain transduction has thrust the identification and characterization of interacting partners that modulate its cellular function. Here, we report that TRPV1 associates with gamma-amino butyric acid A-type (GABA(A)) receptor associated protein (GABARAP) in HEK293 cells and in neurons from dorsal root ganglia coexpressing both proteins. At variance with controls, GABARAP augmented TRPV1 expression in cotransfected cells and stimulated surface receptor clustering. Functionally, GABARAP expression attenuated voltage and capsaicin sensitivity of TRPV1 in the presence of extracellular calcium. Furthermore, the presence of the anchor protein GABARAP notably lengthened the kinetics of vanilloid-induced tachyphylaxia. Notably, the presence of GABARAP selectively increased the interaction of tubulin with the C-terminal domain of TRPV1. Disruption of tubulin cytoskeleton with nocodazole reduced capsaicin-evoked currents in cells expressing TRPV1 and GABARAP, without affecting the kinetics of vanilloid-induced desensitization. Taken together, these findings indicate that GABARAP is an important component of the TRPV1 signaling complex that contributes to increase the channel expression, to traffic and cluster it on the plasma membrane, and to modulate its functional activity at the level of channel gating and desensitization.

Differential contribution of SNARE-dependent exocytosis to inflammatory potentiation of TRPV1 in nociceptors.

Potentiation of the pain-integrator ion channel transient receptor potential vanilloid type 1 (TRPV1) underlies thermal hyperalgesia mediated by a variety of proinflammatory factors. Two complementary mechanisms of TRPV1 inflammatory sensitization have been proposed, namely a decrease of its activation threshold and an increment of its surface expression in nociceptors. Here we investigated the involvement of regulated exocytosis to the inflammatory sensitization of TRPV1 in rat neonatal dorsal root ganglion neurons by proalgesic agents. The contribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent exocytosis was evaluated using a small peptide patterned after the synaptosomal-associated protein of 25 kDa (SNAP-25) protein that acts as a specific and potent inhibitor of neuronal exocytosis. We found that TRPV1 sensitization mediated by nerve growth factor, ATP, and IGF-I was accompanied by a higher channel expression in the neuronal plasma membrane, which was prevented by blockade of regulated exocytosis. In contrast, TRPV1 sensitization caused by bradykinin, IL-1beta, and artemin was insensitive to inhibition of SNARE-dependent vesicular fusion and was not due to an increase in TRPV1 surface expression. Therefore, it appears that some, but not all, proinflammatory agents sensitize rat nociceptors by promoting the recruitment of TRPV1 channels to the neuronal surface. These findings support the tenet that SNARE complex-mediated exocytosis of TRPV1 may be a valid therapeutic target to treat inflammatory pain.

Fast and stable recording of birefringence and holographic gratings in an azo-polymethacrylate using a single nanosecond light pulse.

Pulsed light-induced recording in azobenzene polymers has recently been studied due to its potential use in optical storage applications. In this paper we study the photoinduced birefringence (Deltan) and holographic grating recording in an azobenzene side chain liquid-crystalline polymethacrylate irradiating with a single 4 ns light pulse at 532 nm. For some irradiation conditions, Deltan grows in less than 50 ns reaching an essentially stable value of about 10(-2). Holographic gratings have been registered using intensity and polarization patterns. Fast response and stability, similar to those of Deltan, was observed in the holographic recording process. Both light-induced anisotropy and relief contributions have been found in the case of gratings recorded using intensity patterns, relief being the dominant contribution at high recording energies. Polarization gratings have been recorded using two orthogonally circularly polarized beams. The resultant gratings showed stable efficiencies up to 0.8% (measured at 633 nm in 1-mum-thick films) and no measurable relief was observed.

Monte Carlo dosimetric study of the BEBIG Co-60 HDR source.

Although not as widespread as Ir-192, Co-60 is also available on afterloading equipment devoted to high dose rate brachytherapy, mainly addressed to the treatment of gynaecological lesions. The purpose of this study is to obtain the dosimetric parameters of the Co-60 source used by the BEBIG MultiSource remote afterloader (BEBIG GmbH, Germany) for which there are no dosimetric data available in the literature. The Monte Carlo code GEANT4 has been used to obtain the TG43 parameters and the 2D dose rate table in Cartesian coordinates of the BEBIG Co-60 HDR source. The dose rate constant, radial dose function and anisotropy function have been calculated and are presented in a tabular form as well as a detailed 2D dose rate table in Cartesian coordinates. These dosimetric datasets can be used as input data and to validate the treatment planning system calculations.

Technical note: Monte Carlo derivation of TG-43 dosimetric parameters for radiation therapy resources and 3M Cs-137 sources.

In clinical brachytherapy dosimetry, a detailed dose rate distribution of the radioactive source in water is needed in order to plan for quality treatment. Two Cs-137 sources are considered in this study; the Radiation Therapy Resources 67-800 source (Radiation Therapy Resources Inc., Valencia, CA) and the 3M model 6500/6D6C source. A complete dosimetric dataset for both sources has been obtained by means of the Monte Carlo GEANT4 code. Dose rate distributions are presented in two different ways; following the TG43 formalism and in a 2D rectangular dose rate table. This 2D dose rate table is helpful for the TPS quality control and is fully consistent with the TG43 dose calculation formalism. In this work, several improvements to the previously published data for these sources have been included: the source asymmetries were taken explicitly into account in the MC calculations, TG43 data were derived directly from MC calculations, the data radial range was increased, the angular grid in the anisotropy function was increased, and TG43 data is now consistent with the along and away dose rate table as recommended by the TG43 update.

Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation of a single tyrosine residue.

The catalytic domain of clostridial neurotoxins is a substrate of tyrosine-specific protein kinases. The functional role of tyrosine phosphorylation and also the number and location of its (their) phosphorylation site(s) are yet elusive. We have used the recombinant catalytic domain of botulinum neurotoxin E (BoNT E) to examine these issues. Bacterially expressed and purified BoNT E catalytic domain was fully active, and was phosphorylated in vitro by the tyrosine-specific kinase Src. Tyrosine phosphorylation of the catalytic domain increased the protein thermal stability without affecting its proteolytic activity. Covalent modification of the endopeptidase promoted a disorder-to-order transition, as evidenced by the 35% increment of the alpha-helical content, which resulted in a 4 degrees C increase of its denaturation temperature. Site-directed replacement of tyrosine at position 67 completely abolished phosphate incorporation by Src. Constitutively unphosphorylated endopeptidase mutants exhibited functional properties virtually identical to those displayed by the nonphosphorylated wild-type catalytic domain. These findings indicate the presence of a single phosphorylation site in the catalytic domain of clostridial neurotoxins, and that its covalent modification primarily modulates the protein thermostability.

Structural compatibility between the putative voltage sensor of voltage-gated K+ channels and the prokaryotic KcsA channel.

Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K(+) channel (KcsA), support the tenet that voltage-gated K(+) channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.

Arginine-rich peptides are blockers of VR-1 channels with analgesic activity.

Vanilloid receptors (VRs) play a fundamental role in the transduction of peripheral tissue injury and/or inflammation responses. Molecules that antagonize VR channel activity may act as selective and potent analgesics. We report that synthetic arginine-rich hexapeptides block heterologously expressed VR-1 channels with submicromolar efficacy in a weak voltage-dependent manner, consistent with a binding site located near/at the entryway of the aqueous pore. Dynorphins, natural arginine-rich peptides, also blocked VR-1 activity with micromolar affinity. Notably, synthetic and natural arginine-rich peptides attenuated the ocular irritation produced by topical capsaicin application onto the eyes of experimental animals. Taken together, our results imply that arginine-rich peptides are VR-1 channel blockers with analgesic activity. These findings may expand the development of novel analgesics by targeting receptor sites distinct from the capsaicin binding site.

Identification of an aspartic residue in the P-loop of the vanilloid receptor that modulates pore properties.

Vanilloid receptor subunit 1 (VR1) is a nonselective cation channel that integrates multiple pain-producing stimuli. VR1 channels are blocked with high efficacy by the well established noncompetitive antagonist ruthenium red and exhibit high permeability to divalent cations. The molecular determinants that define these functional properties remain elusive. We have addressed this question and evaluated by site-specific neutralization the contribution on pore properties of acidic residues located in the putative VR1 pore region. Mutant receptors expressed in Xenopus oocytes exhibited capsaicin-operated ionic currents akin to those of wild type channels. Incorporation of glutamine residues at Glu(648) and Glu(651) rendered minor effects on VR1 pore attributes, while Glu(636) slightly modulated pore blockade. In contrast, replacement of Asp(646) by asparagine decreased 10-fold ruthenium red blockade efficacy and reduced 4-fold the relative permeability of the divalent cation Mg(2+) with respect to Na(+) without changing the selectivity of monovalent cations. At variance with wild type channels and E636Q, E648Q, and E651Q mutant receptors, ruthenium red blockade of D646N mutants was weakly sensitive to extracellular pH acidification. Collectively, our results suggest that Asp(646) is a molecular determinant of VR1 pore properties and imply that this residue may form a ring of negative charges that structures a high affinity binding site for cationic molecules at the extracellular entryway.

Neuronal death and perinatal lethality in voltage-gated sodium channel alpha(II)-deficient mice.

Neural activity is crucial for cell survival and fine patterning of neuronal connectivity during neurodevelopment. To investigate the role in vivo of sodium channels (NaCh) in these processes, we generated knockout mice deficient in brain NaChalpha(II). NaChalpha(II)(-/-) mice were morphologically and organogenically indistinguishable from their NaChalpha(+/-) littermates. Notwithstanding, NaChalpha(II)(-/-) mice died perinatally with severe hypoxia and massive neuronal apoptosis, notably in the brainstem. Sodium channel currents recorded from cultured neurons of NaChalpha(II)(-/-) mice were sharply attenuated. Death appears to arise from severe hypoxia consequent to the brainstem deficiency of NaChalpha(II). NaChalpha(II) expression is, therefore, redundant for embryonic development but essential for postnatal survival.

Structural and functional modularity of voltage-gated potassium channels.

Sequence similarity among known potassium channels indicates the voltage-gated potassium channels consist of two modules: the N-terminal portion of the channel up to and including transmembrane segment S4, called in this paper the 'sensor' module, and the C-terminal portion from transmembrane segment S5 onwards, called the 'pore' module. We investigated the functional role of these modules by constructing chimeric channels which combine the 'sensor' from one native voltage-gated channel, mKv1.1, with the 'pore' from another, Shaker H4, and vice versa. Functional studies of the wild type and chimeric channels show that these modules can operate outside their native context. Each channel has a unique conductance-voltage relation. Channels incorporating the mKv1.1 sensor module have similar rates of activation while channels having the Shaker pore module show similar rates of deactivation. This observation suggests the mKv1.1 sensor module limits activation and the Shaker pore module determines deactivation. We propose a model that explains the observed equilibrium and kinetic properties of the chimeric constructs in terms of the characteristics of the native modules and a novel type of intrasubunit cooperativity. The properties ascribed to the modules are the same whether the modules function in their native context or have been assembled into a chimera.

Structural determinants of the blocker binding site in glutamate and NMDA receptor channels.

Glutamate receptor channels of the NMDA-type (N-methyl-D-aspartate) and non-NMDA-type (GluR) differ in their pore properties. The N-site in the M2 transmembrane segment of NMDA receptors (NMDAR), or the corresponding Q/R-site in GluRs, is a pivotal structural determinant of their permeation and blockade characteristics. Substitutions at a second site in M2, the L-site (L577) in GluR1, drastically alter the receptor selectivity to divalent cations. Here we report that M2 mutants carrying an asparagine or a threonine residue at the Q-site of GluR1, along with a tryptophan residue at the L-site, form homomeric GluR1 channels that are highly sensitive to structurally diverse, uncompetitive NMDA antagonists such as arylcyclohexylamines, dibenzocycloheptenimines, and to morphinian and adamantane derivatives. Analysis of the voltage dependence of channel blockade locates the blocker binding site approximately 0.65 partway into the transmembrane electric field in both GluR1 mutants and NMDAR channels. Our results suggest that the homomeric GluR1 double mutants, L577W/Q582N and L577W/Q582T, fairly approximate the pore properties of the heteromeric NMDA receptor and support the structural kinship of their permeation pathways.

Mutation of conserved negatively charged residues in the S2 and S3 transmembrane segments of a mammalian K+ channel selectively modulates channel gating.

Voltage-gated channel proteins sense a change in the transmembrane electric field and respond with a conformational change that allows ions to diffuse across the pore-forming structure. Site-specific mutagenesis combined with electrophysiological analysis of expressed mutants in amphibian oocytes has previously established the S4 transmembrane segment as an element of the voltage sensor. Here, we show that mutations of conserved negatively charged residues in S2 and S3 of a brain K+ channel, thought of as countercharges for the positively charged residues in S4, selectively modulate channel gating without modifying the permeation properties. Mutations of Glu235 in S2 that neutralize or reverse charge increase the probability of channel opening and the apparent gating valence. In contrast, replacements of Glu272 by Arg or Thr268 by Asp in S3 decrease the open probability and the apparent gating valence. Residue Glu225 in S2 tolerated replacement only by acidic residues, whereas Asp258 in S3 was intolerant to any attempted change. These results imply that S2 and S3 are unlikely to be involved in channel lining, yet, together with S4, may be additional components of the voltage-sensing structure.

Detergent-induced reversible denaturation of the photosystem II reaction center: implications for pigment-protein interactions.

Incubation of the D1-D2-cytochrome b559 complex with Triton X-100 modified the protein secondary structure, caused significant spectral modifications, and reduced the formation of light-induced spin-polarized triplet electron paramagnetic resonance (EPR) signal. After 24 h of incubation, the absorption spectrum shifted from 675.5 to 671.5 nm and the fluorescence spectrum shifted from 682 to 672 nm. These shifts were accompanied by an increase in the chlorophyll fluorescence yield and by decreases in the intensity of the circular dichroism in the red region and the secondary electron transport activity. The intensity of the light-induced triplet EPR signal was also markedly reduced in the same experimental conditions. Substitution of dodecyl beta-maltoside for Triton X-100 reversed all the above-mentioned parameters to the values exhibited by the native D1-D2-Cyt b559 complex, including the characteristic triplet EPR signal. We concluded that all observed changes were due to the destruction of P680 with Triton X-100 and to the reestablishment of P680 in the presence of dodecyl beta-maltoside. The easier but certainly not the only possible explanation to all these phenomena is to consider a dimeric structure for P680, at least in its ground state, where interactions take place within the two dimeric chromophores and with the apoprotein. Such a dimeric structure would be very sensitive to small modifications of the P680 domain, which convert the dimer absorbing at 680 into two chlorophyll monomers absorbing near 670 nm. The dodecyl beta-maltoside reestablished the structure of the native P680 domain.

Core antenna complexes, CP43 and CP47, of higher plant photosystem II. Spectral properties, pigment stoichiometry, and amino acid composition.

The core antenna complexes of photosystem II, CP43 and CP47, were purified from two higher plants by anion-exchange chromatography, using a combination of the chaotropic agent LiClO4 and the nonionic detergent beta-dodecyl maltoside. The Qy transition was resolved at 48 K into two main bands near 682.3 and 671.5 nm for CP43, while the CP47 spectrum showed a more complex structure with main bands at 688, 681.2, 676, 667, and 661 nm. Emission bands (77 K) were detected at 683 and 695 nm for CP43 and CP47, respectively. Fluorescence excitation spectra showed high efficiency of energy transfer between the different transitions of the chlorophylls and a somewhat lower efficiency from beta-carotene. The circular dichroism spectrum of CP47 indicated the presence of excitonic interactions between some chlorophylls. In contrast, CP43 showed a single negative circular dichroism band at 670 nm. The pigment content of the complexes was determined by both spectroscopic measurements and HPLC. Contents of 18 chlorophylls a and 5 beta-carotenes per CP43 polypeptide and 19 chlorophylls a and 3 beta-carotenes per CP47 polypeptide were found, using the methods of Lowry or Bradford for protein quantitation. When the protein concentration was determined from the amino acid analysis, 20 chlorophylls a and 5 beta-carotenes per CP43 and 21-22 chlorophylls a and 4 beta-carotenes per CP47 were obtained. Thus, a content of 46-48 chlorophylls a was obtained for the core complex, assuming 4-6 chlorophylls per reaction center, in agreement with the composition obtained experimentally using a highly purified oxygen-evolving core complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively.

A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor subunit (NMDAR1) and a proposed glutamate-binding subunit of the NMDA receptor (NMDARA1) have provided an opportunity to test the hypothesis that either of these genes might be directly involved in the causation of HD. We have mapped NMDAR1 to 9q34.3 using in situ hybridization studies and NMDARA1 to human chromosome 8 using a somatic cell hybrid panel. Because the gene causing HD has been localized to chromosome 4p16.3, the chromosome assignments reported here are inconsistent with either of these genes playing a causative role in the molecular pathology of HD. However, it is noteworthy that the gene for torsion dystonia has also been localized by genetic studies to 9q34.3, the same regional map location as NMDAR1.

Molecular cloning, functional expression, and pharmacological characterization of an N-methyl-D-aspartate receptor subunit from human brain.

A cDNA encoding a full-length N-methyl-D-aspartate (NMDA) receptor subunit 1, hNR1, was isolated from a human brain cDNA library. The hNR1 cDNA encodes an open reading frame of approximately 2.7 kb that shares high homology with the rat brain NMDA receptor subunit 1 and the mouse zeta 1 subunit. The hNR1 sequence, however, diverges from the rodent and murine homologs near the C terminus, suggesting that they represent alternatively spliced messages of the same gene. Oocytes injected with cRNA synthesized from the hNR1 cDNA express glutamate and NMDA-activated currents in the presence of glycine. Currents are blocked by the NMDA-receptor-specific antagonists 2-amino-5-phosphovaleric acid and 7-chlorokynurenate, and the open channel blockers MK-801 and phencyclidine, by Mg2+ ions in a voltage-dependent manner, and by Zn2+. Expressed hNR1 homomeric receptor channels exhibit the high Ca2+ permeability characteristic of neuronal NMDA receptors. Therefore, the cDNA clone hNR1 codes for a human brain NMDA receptor subunit cognate to the rodent and murine brain NR1 subunits.

Possible coexistence of two independent mechanisms contributing to anthracycline resistance in leukaemia P388 cells.

Murine leukaemia P388 and L1210 cell sublines with varying degrees of resistance to the anthracycline daunomycin (DNM) have been used to monitor (i) intracellular accumulation of DNM, (ii) expression of the drug efflux pump P-glycoprotein (pgp) and (iii) cytoplasmic pH changes. Drug-resistant L1210/65 cells (65-fold resistance), overexpress pgp, and display decreased intracellular accumulation of DNM and identical intracellular pH as compared to the parental drug-sensitive L1210 cell line. On the other hand, moderately drug-resistant P388/20 cells (20-fold resistance), which also exhibit a decreased intracellular drug accumulation with respect to drug-sensitive P388/S cells, display only moderate pgp-encoding mdr1 gene transcription without detectable levels of pgp protein, and undergo cytoplasmic alkalinisation (up to approximately 0.2 pH units). A further increase in the level of drug resistance (P388/100 cells, 100-fold resistance), results in a more pronounced decrease in drug accumulation, significant pgp expression and slightly higher intracellular alkalinisation. Alterations in the degree of protonation of DNM have been shown previously to influence processes such as the rate of uptake and the intracellular accumulation of the drug. On this basis, we propose that the changes in intracellular pH, observed at low levels of drug resistance (P388/20 cells), could constitute an early cellular response aimed at decreasing the intracellular accumulation of ionisable anti-neoplastics. As the level of resistance increases (P388/100), the cells seem to require more efficient mechanisms of defense against the drug, such as that represented by the expression of pgp. Since there is no apparent correlation between the extent of the changes in intracellular pH and the level of pgp expression in DNM-resistant P388 cell sublines, it is suggested that these two cellular responses contributing to drug resistance could operate independently.

Verapamil reverses the ultrastructural alterations in the plasma membrane induced by drug resistance.

Two P388 cell sublines with different levels of resistance to daunomycin (DNM), P388/20 and P388/100 cells (approximately 20- and 100-fold resistance, respectively), undergo a significant (approximately 2-fold) increase in the number of intramembrane particles (IMPs) present at their plasma membrane, as compared to that exhibited by the parental, drug-sensitive P388 (P388/S) cell line. Regardless of the level of resistance, incubation of drug-resistant cells with verapamil, a well known reverting agent of anthracycline resistance, restores the morphology of the plasma membrane in these cells, yielding a pattern in which the number and size distribution of IMPs at both leaflets of the bilayer, become undistinguishable from those displayed by drug-sensitive cells. Furthermore, verapamil did not affect the ultrastructural organization of the plasma membrane of drug-sensitive cells. It is possible that the alterations in the structural organization of the plasma membrane of the antineoplastic-resistant tumor cells, might represent a reliable 'marker' for early diagnosis of drug resistance.