Endogenous, cholesterol-activated ATP-dependent transport in membrane vesicles from Spodoptera frugiperda cells

Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda -derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,′7′-dichlorofluorescein (CDCF), estradiol-17β-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 ± 1.11 μM. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda-derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,' 7'-dichlorofluorescein (CDCF), estradiol-17 beta-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 +/- 1.11 mu M. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.Peer reviewe

Association of CSF proteins with tau and amyloid β levels in asymptomatic 70-year-olds

BACKGROUND: Increased knowledge of the evolution of molecular changes in neurodegenerative disorders such as Alzheimer's disease (AD) is important for the understanding of disease pathophysiology and also crucial to be able to identify and validate disease biomarkers. While several biological changes that occur early in the disease development have already been recognized, the need for further characterization of the pathophysiological mechanisms behind AD still remains. METHODS: In this study, we investigated cerebrospinal fluid (CSF) levels of 104 proteins in 307 asymptomatic 70-year-olds from the H70 Gothenburg Birth Cohort Studies using a multiplexed antibody- and bead-based technology. RESULTS: The protein levels were first correlated with the core AD CSF biomarker concentrations of total tau, phospho-tau and amyloid beta (Aβ42) in all individuals. Sixty-three proteins showed significant correlations to either total tau, phospho-tau or Aβ42. Thereafter, individuals were divided based on CSF Aβ42/Aβ40 ratio and Clinical Dementia Rating (CDR) score to determine if early changes in pathology and cognition had an effect on the correlations. We compared the associations of the analysed proteins with CSF markers between groups and found 33 proteins displaying significantly different associations for amyloid-positive individuals and amyloid-negative individuals, as defined by the CSF Aβ42/Aβ40 ratio. No differences in the associations could be seen for individuals divided by CDR score. CONCLUSIONS: We identified a series of transmembrane proteins, proteins associated with or anchored to the plasma membrane, and proteins involved in or connected to synaptic vesicle transport to be associated with CSF biomarkers of amyloid and tau pathology in AD. Further studies are needed to explore these proteins' role in AD pathophysiology

Origin of the photoemission final-state effects in Bi2Sr2CaCu2O8 by very-low-energy electron diffraction

Very-low-energy electron diffraction with a support of full-potential band calculations is used to achieve the energy positions, K// dispersions, lifetimes and Fourier compositions of the photoemission final states in Bi2Sr2CaCu2O8 at low excitation energies. Highly structured final states explain the dramatic matrix element effects in photoemission. Intense c(2x2) diffraction reveals a significant extrinsic contribution to the shadow Fermi surface. The final-state diffraction effects can be utilized to tune the photoemission experiment on specific valence states or Fermi surface replicas.Comment: 4 pages, 3 Postscript figures, submitted to Phys. Rev. Lett; major revision

First Principles Calculations of Fe on GaAs (100)

We have calculated from first principles the electronic structure of 0.5 monolayer upto 5 monolayer thick Fe layers on top of a GaAs (100) surface. We find the Fe magnetic moment to be determined by the Fe-As distance. As segregates to the top of the Fe film, whereas Ga most likely is found within the Fe film. Moreover, we find an asymmetric in-plane contraction of our unit-cell along with an expansion perpendicular to the surface. We predict the number of Fe 3d-holes to increase with increasing Fe thickness on $p$-doped GaAs.Comment: 9 pages, 14 figures, submitted to PR

Identification of Genes Contributing to the Virulence of Francisella tularensis SCHU S4 in a Mouse Intradermal Infection Model

Background: Francisella tularensis is a highly virulent human pathogen. The most virulent strains belong to subspecies tularensis and these strains cause a sometimes fatal disease. Despite an intense recent research effort, there is very limited information available that explains the unique features of subspecies tularensis strains that distinguish them from other F. tularensis strains and that explain their high virulence. Here we report the use of targeted mutagenesis to investigate the roles of various genes or pathways for the virulence of strain SCHU S4, the type strain of subspecies tularensis. Methodology/Principal Findings: The virulence of SCHU S4 mutants was assessed by following the outcome of infection after intradermal administration of graded doses of bacteria. By this route, the LD\u2085\u2080 of the SCHU S4 strain is one CFU. The virulence of 20 in-frame deletion mutants and 37 transposon mutants was assessed. A majority of the mutants did not show increased prolonged time to death, among them notably \u394pyrB and \u394recA. Of the remaining, mutations in six unique targets, tolC, rep, FTT0609, FTT1149c, ahpC, and hfq resulted in significantly prolonged time to death and mutations in nine targets, rplA, wbtI, iglB, iglD, purL, purF, ggt, kdtA, and glpX, led to marked attenuation with an LD\u2085\u2080 of >10\ub3 CFU. In fact, the latter seven mutants showed very marked attenuation with an LD\u2085\u2080 of 6510\u2077 CFU. Conclusions/Significance: The results demonstrate that the characterization of targeted mutants yielded important information about essential virulence determinants that will help to identify the so far little understood extreme virulence of F. tularensis subspecies tularensis.Peer reviewed: YesNRC publication: Ye

Study of the pressure effects in TiOCl by ab initio calculations

Electronic structure calculations on the low dimensional spin-1/2 compound TiOCl were performed at several pressures in the orthorhombic phase, finding that the structure is quasi-one-dimensional. The Ti3+ (d1) ions have one t2g orbital occupied (dyz) with a large hopping integral along the b direction of the crystal. The most important magnetic coupling is Ti-Ti along the b axis. The transition temperature (Tc) has a linear evolution with pressure, and at about 10 GPa this Tc is close to room temperature, leading to a room temperature spin-Peierls insulator-insulator transition, with an important reduction of the charge gap in agreement with the experiment. On the high-pressure monoclinic phase, TiOCl presents two possible dimerized structures, with a long or short dimerization. Long dimerized state occurs above 15 GPa, and below this pressure the short dimerized structure is the more stable phase.Comment: 3 pages, 3 embedded figures, 1 table. A. Pi\~neiro, et al.,J. Magn. Magn. Mater. (2009) (accepted

Evidence of Strong Electron Correlations in Gamma-Iron

Single-particle excitation spectra of gamma-Fe in the paramagnetic state have been investigated by means of the first-principles dynamical coherent potential approximation theory which has recently been developed. It is found that the central peak in the density of states consisting of the t2g bands is destroyed by electron correlations, and the Mott-Hubbard type correlated bands appear. The results indicate that the gamma-Fe can behave as correlated electrons at high temperatures.Comment: 7 pages, 3 figures, to be published in J. Phys. Soc. Jpn. Vol.78, No.9 (2009