Exploration of new drug delivery pathways: I. Mechanism of folate uptake in cultured human cells. II. Role of nuclear localization signal peptides in the delivery of oligonucleotide into reconstituted nuclei

The roles of the folate receptor and an anion carrier in the uptake of 5- methyltetrahydrofolate (5-MeH_4folate) were studied in cultured human (KB) cells using radioactive 5-MeH_4folate. Binding of the 5-MeH_4folate was inhibited by folic acid, but not by probenecid, an anion carrier inhibitor. The internalization of 5-MeH_4folate was inhibited by low temperature, folic acid, probenecid and methotrexate. Prolonged incubation of cells in the presence of high concentrations of probenecid appeared to inhibit endocytosis of folatereceptors as well as the anion carrier. The V_(max) and K_M values for the carrier were 8.65 ± 0.55 pmol/min/mg cell protein and 3.74 ± 0.54µM, respectively. The transport of 5-MeH4folate was competitively inhibited by folic acid, probenecid and methotrexate. The carrier dissociation constants for folic acid, probenecid and methotreate were 641 µM, 2.23 mM and 13.8 µM, respectively. Kinetic analysis suggests that 5-MeH_4folate at physiological concentration is transported through an anion carrier with the characteristics of the reduced-folate carrier after 5-MeH_4folate is endocytosed by folate receptors in KB cells. Our data with KB cells suggest that folate receptors and probenecid-sensitive carriers work in tandem to transport 5-MeH_4folate to the cytoplasm of cells, based upon the assumption that 1 mM probenecid does not interfere with the acidification of the vesicle where the folate receptors are endocytosed.

Oligodeoxynucleotides designed to hybridize to specific mRNA sequences (antisense oligonucleotides) or double stranded DNA sequences have been used to inhibit the synthesis of a number of cellular and viral proteins (Crooke, S. T. (1993) FASEB J. 7, 533-539; Carter, G. and Lemoine, N. R. (1993) Br. J. Cacer 67, 869-876; Stein, C. A. and cohen, J. S. (1988) Cancer Res. 48, 2659-2668). However, the distribution of the delivered oligonucleotides in the cell, i.e., in the cytoplasm or in the nucleus has not been clearly defined. We studied the kinetics of oligonucleotide transport into the cell nucleus using reconstituted cell nuclei as a model system. We present evidences here that oligonucleotides can freely diffuse into reconstituted nuclei. Our results are consistent with the reports by Leonetti et al. (Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 2702-2706, April 1991), which were published while we were carrying this research independently. We also investigated whether a synthetic nuclear localization signal (NLS) peptide of SV40 T antigen could be used for the nuclear targeting of oligonucleotides. We synthesized a nuclear localization signal peptide-conjugated oligonucleotide to see if a nuclear localization signal peptide can enhance the uptake of oligonucleotides into reconstituted nuclei of Xenopus. Uptake of the NLS peptide-conjugated oligonucleotide was comparable to the control oligonucleotide at similar concentrations, suggesting that the NLS signal peptide does not significantly enhance the nuclear accumulation of oligonucleotides. This result is probably due to the small size of the oligonucleotide.

Polaron hopping in olivine phosphates studied by nuclear resonant scattering

Valence fluctuations of Fe²⁺ and Fe³⁺ were studied in a solid solution of Li_xFePO₄ by nuclear resonant forward scattering of synchrotron x rays while the sample was heated in a diamond-anvil pressure cell. The spectra acquired at different temperatures and pressures were analyzed for the frequencies of valence changes using the Blume-Tjon model of a system with a fluctuating Hamil- tonian. These frequencies were analyzed to obtain activation energies and an activation volume for polaron hopping. There was a large suppression of hopping frequency with pressure, giving an anomalously large activation volume. This large, positive value is typical of ion diffusion, which indicates correlated motions of polarons, and Li⁺ ions that alter the dynamics of both.

In a parallel study of Na_xFePO₄, the interplay between sodium ordering and electron mobility was investigated using a combination of synchrotron x-ray diffraction and nuclear resonant scattering. Conventional Mossbauer spectra were collected while the sample was heated in a resistive furnace. An analysis of the temperature evolution of the spectral shapes was used to identify the onset of fast electron hopping and determine the polaron hopping rate. Synchrotron x-ray diffraction measurements were carried out in the same temperature range. Reitveld analysis of the diffraction patterns was used to determine the temperature of sodium redistribution on the lattice. The diffraction analysis also provides new information about the phase stability of the system. The temperature evolution of the iron site occupancies from the Mossbauer measurements, combined with the synchrotron diffraction results give strong evidence for a relationship between the onset of fast electron dynamics and the redistribution of sodium in the lattice.

Measurements of activation barriers for polaron hopping gave fundamental insights about the correlation between electronic carriers and mobile ions. This work established that polaron-ion interactions can alter the local dynamics of electron and ion transport. These types of coupled processes may be common in many materials used for battery electrodes, and new details concerning the influence of polaron-ion interactions on the charge dynamics are relevant to optimizing their electrochemical performance.

Simulações de problemas inversos com aplicações em engenharia nuclear usando técnicas de transporte de partículas neutras monoenergéticas na formulação unidimensional de ordenadas discretas

Neste trabalho, três técnicas para resolver numericamente problemas inversos de transporte de partículas neutras a uma velocidade para aplicações em engenharia nuclear são desenvolvidas. É fato conhecido que problemas diretos estacionários e monoenergéticos de transporte são caracterizados por estimar o fluxo de partículas como uma função-distribuição das variáveis independentes de espaço e de direção de movimento, quando os parâmetros materiais (seções de choque macroscópicas), a geometria, e o fluxo incidente nos contornos do domínio (condições de contorno), bem como a distribuição de fonte interior são conhecidos. Por outro lado, problemas inversos, neste trabalho, buscam estimativas para o fluxo incidente no contorno, ou a fonte interior, ou frações vazio em barras homogêneas. O modelo matemático usado tanto para os problemas diretos como para os problemas inversos é a equação de transporte independente do tempo, a uma velocidade, em geometria unidimensional e com o espalhamento linearmente anisotrópico na formulação de ordenadas discretas (SN). Nos problemas inversos de valor de contorno, dado o fluxo emergente em um extremo da barra, medido por um detector de nêutrons, por exemplo, buscamos uma estimativa precisa para o fluxo incidente no extremo oposto. Por outro lado, nos problemas inversos SN de fonte interior, buscamos uma estimativa precisa para a fonte armazenada no interior do domínio para fins de blindagem, sendo dado o fluxo emergente no contorno da barra. Além disso, nos problemas inversos SN de fração de vazio, dado o fluxo emergente em uma fronteira da barra devido ao fluxo incidente prescrito no extremo oposto, procuramos por uma estimativa precisa da fração de vazio no interior da barra, no contexto de ensaios não-destrutivos para aplicações na indústria. O código computacional desenvolvido neste trabalho apresenta o método espectronodal de malha grossa spectral Greens function (SGF) para os problemas diretos SN em geometria unidimensional para gerar soluções numéricas precisas para os três problemas inversos SN descritos acima. Para os problemas inversos SN de valor de contorno e de fonte interior, usamos a propriedade da proporcionalidade da fuga de partículas; ademais, para os problemas inversos SN de fração de vazio, oferecemos a técnica a qual nos referimos como o método físico da bissecção. Apresentamos resultados numéricos para ilustrar a precisão das três técnicas, conforme descrito nesta tese.

Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

Assessment of siltation rate and sediment transport in Bombay harbour based on Cesium-137 distribution

Low level aqueous radioactive wastes from the nuclear complex at Trombay are discharged into the Bombay harbour. Of the 6 principal radionuclides comprising the discharge, cesium-137 is most predominantly taken up by silt from the sea water. Isoactivity contours show that activity levels just off the point of discharge, and further south to Pir Pau, were between 50 and 100 pCi/g. Activity in the main channel of the harbour remained below 10 pCi/g, while further north (below the Thane Creek bridge) it was around 20 pCi/g. Activity in the top 6 cm of a core off Trombay naval jetty was <5 pCi/g, reaching a maximum of 178 pCi/g at 14cm below the surface. Thereafter, it tapered off to 5 pCi/g down to a metre.

Circumstantial Evidence for a Soft Nuclear Symmetry Energy at Suprasaturation Densities

Within an isospin- and momentum-dependent hadronic transport model, it is shown that the recent FOPI data on the pi(-)/pi(+) ratio in central heavy-ion collisions at SIS/GSI energies [Willy Reisdorf , Nucl. Phys. A 781, 459 (2007)] provide circumstantial evidence suggesting a rather soft nuclear symmetry energy E-sym(rho) at rho >= 2 rho(0) compared to the Akmal-Pandharipande-Ravenhall prediction. Some astrophysical implications and the need for further experimental confirmations are discussed.

Triton-He-3 relative and differential flows as probes of the nuclear symmetry energy at supra-saturation densities

Using a transport model coupled with a phase-space coalescence afterburner, we study the triton-He-3 (t-He-3) ratio with both relative and differential transverse flows in semicentral Sn-132 + Sn-124 reactions at a beam energy of 400 MeV/nucleon. The neutron-proton ratios with relative and differential flows are also discussed as a reference. We find that similar to the neutron-proton pairs, the t-He-3 pairs also carry interesting information regarding the density dependence of the nuclear symmetry energy. Moreover, the nuclear symmetry energy affects more strongly the t-He-3 relative and differential flows than the pi(-)/pi(+) ratio in the same reaction. The t-He-3 relative flow can be used as a particularly powerful probe of the high-density behavior of the nuclear symmetry energy.

Neutron-proton bremsstrahlung from intermediate energy heavy-ion reactions as a probe of the nuclear symmetry energy?

Hard photons from neutron-proton bremsstrahlung in intermediate energy heavy-ion reactions are examined as a potential probe of the nuclear symmetry energy within a transport model. Effects of the symmetry energy on the yields and spectra of hard photons are found to be generally smaller than those due to the currently existing uncertainties of both the in-medium nucleon-nucleon cross sections and the photon production probability in the elementary process pn -> pn gamma. Very interestingly, nevertheless, the ratio of hard photon spectra R-1/2(gamma) from two reactions using isotopes of the same element is not only approximately independent of these uncertainties but also quite sensitive to the symmetry energy. For the head-on reactions of Sn-132 + Sn-124 and Sn-112 + Sn-112 at E-beam/A = 50 MeV, for example, the R-1/2(gamma) displays a rise up to 15% when the symmetry energy is reduced by about 20% at rho = 1.3 rho(0) which is the maximum density reached in these reactions. (C) 2008 Elsevier B.V. All rights reserved.

Differential isospin-fractionation in dilute asymmetric nuclear matter

The differential isospin-fractionation (IsoF) during the liquid-gas phase transition in dilute asymmetric nuclear matter is studied as a function of nucleon momentum. Within a self-consistent thermal model it is shown that the neutron/proton ratio of the gas phase becomes smaller than that of the liquid phase for energetic nucleons, although the gas phase is overall more neutron-rich. Clear indications of the differential IsoF consistent with the thermal model predictions are demonstrated within a transport model for heavy-ion reactions. Future comparisons with experimental data will allow us to extract critical information about the momentum dependence of the isovector strong interaction.

The neutron/proton ratio of squeezed-out nucleons and the high density behavior of the nuclear symmetry energy

Within a transport model it is shown that the neutron/proton ratio of squeezed-out nucleons perpendicular to the reaction plane, especially at high transverse momenta, in heavy-ion reactions induced by high energy neutron-rich nuclei can be a useful tool for studying the high density behavior of the nuclear symmetry energy.

Double neutron-proton differential transverse flow as a probe for the high density behavior of the nuclear symmetry energy

The double neutron-proton differential transverse flow taken from two reaction systems using different isotopes of the same element is studied at incident beam energies of 400 and 800 MeV/nucleon within the framework of an isospin- and momentum-dependent hadronic transport model IBUU04. The double differential flow is found to retain about the same sensitivity to the density dependence of the nuclear symmetry energy as the single differential flow in the more neutron-rich reaction. Because the double differential flow reduces significantly both the systematic errors and the influence of the Coulomb force, it is thus more effective probe for the high-density behavior of the nuclear symmetry energy.

Single and double pi(-)/pi(+) ratios in heavy-ion reactions as probes of the high-density behavior of the nuclear symmetry energy

Based on the isospin- and momentum-dependent hadronic transport model IBUU04, effects of the nuclear symmetry energy on the single and double pi(-)/pi(+) ratios in central reactions of Sn-132+Sn-124 and Sn-112+Sn-112 at a beam energy of 400 MeV/nucleon are studied. It is found that around the Coulomb peak of the single pi(-)/pi(+) ratio the double pi(-)/pi(+) ratio taken from the two isotopic reactions retains about the same sensitivity to the density dependence of nuclear symmetry energy. Because the double pi(-)/pi(+) ratio can significantly reduce the systematic errors, it is thus a more effective probe for the high-density behavior of the nuclear symmetry energy.

Double neutron/proton ratio of nucleon emissions in isotopic reaction systems as a robust probe of nuclear symmetry energy

The double neutron/proton ratio of nucleon emissions taken from two reaction systems using four isotopes of the same element, namely, the neutron/proton ratio in the neutron-rich system over that in the more symmetric system, has the advantage of reducing systematically the influence of the Coulomb force and the normally poor efficiencies of detecting low energy neutrons. The double ratio thus suffers less systematic errors. Within the IBUU04 transport model the double neutron/proton ratio is shown to have about the same sensitivity to the density dependence of nuclear symmetry energy as the single neutron/proton ratio in the neutron-rich system involved. The double neutron/proton ratio is therefore more useful for further constraining the symmetry energy of neutron-rich matter.

System size and beam energy effects on probing the high-density behavior of nuclear symmetry energy with pion ratio

Based on the isospin-and momentum-dependent hadronic transport model IBUU04, we have investigated the pi(-)/pi(+) ratio in the following three reactions: Ca-48+Ca-48, Sn-124 +Sn-124 and Au-197+Au-197 with nearly the same isospin asymmetry but different masses, at the bombarding energies from 0.25 to 0.6 A GeV. It is shown that the sensitivity of probing the E-sym (rho) with pi(-)/pi(+) increases with increasing the system size or decreasing the beam energy, showing a correlation to the degree of isospin fractionation. Therefore, with a given isospin asymmetry, heavier system at energies near the pion threshold is preferential to study the behavior Of nuclear symmetry energy at supra-saturation densities.