Impact of metal on the DNA photocleavage activity and cytotoxicity of ferrocenyl terpyridine 3d metal complexes

Ferrocenyl terpyridine 3d metal complexes and their analogues, viz. [M(Fc-tpy)2](ClO4)2 (1-4), [Zn(Ph-tpy)2](ClO4)2 (5) and [Zn(Fc-dpa)2]X2 (X = ClO4, 6; PF6, 6a), where M = Fe(II) in 1, Co(II) in 2, Cu(II) in 3 and Zn(II) in 4, Fc-tpy is 4'-ferrocenyl-2,2':6',2''-terpyridine, Ph-tpy is 4'-phenyl-2,2':6',2''-terpyridine and Fc-dpa is ferrocenyl-N,N-dipicolylmethanamine, are prepared and their DNA binding and photocleavage activity in visible light studied. Complexes 2, 4, 5 and 6a that are structurally characterized by X-ray crystallography show distorted octahedral geometry with the terpyridyl ligands binding to the metal in a meridional fashion, with Fc-dpa in 6a showing a facial binding mode. The Fc-tpy complexes display a charge transfer band in the visible region. The ferrocenyl (Fc) complexes show a quasi-reversible Fc+-Fc redox couple within 0.48 to 0.66 V vs. SCE in DMF-0.1 M TBAP. The DNA binding constants of the complexes are 104 M-1. Thermal denaturation and viscometric data suggest DNA surface binding through electrostatic interaction by the positively charged complexes. Barring the Cu(II) complex 3, the complexes do not show any chemical nuclease activity in the presence of glutathione. Complexes 1-4 exhibit significant plasmid DNA photocleavage activity in visible light via a photoredox pathway. Complex 5, without the Fc moiety, does not show any DNA photocleavage activity. The Zn(II) complex 4 shows a significant PDT effect in HeLa cancer cells giving an IC50 value of 7.5 µ M in visible light, while being less toxic in the dark (IC50 = 49 µ M)

C-terminal Tail of β-Tubulin and its Role in the Alterations of Dynein Binding Mode

Dynein is a cytoskeletal molecular motor protein that moves along the microtubule (MT) and transports various cellular cargos during its movement. Using standard Molecular Dynamics (MD) simulation, Principle Component Analysis (PCA), and Normal Mode Analysis (NMA) methods, this investigation studied large-scale movements and local interactions of dynein’s Microtubule Binding Domain (MTBD) when bound to tubulin heterodimer subunits. Examination of the interactions between the MTBD segments, and their adjustments in terms of intra- and intermolecular distances at the interfacial area with tubulin heterodimer, particularly at α-H16, β-H18 and β-tubulin C-terminal tail (CTT), was the main focus of this study. The specific intramolecular interactions, electrostatic forces and the salt-bridge residue pairs were shown to be the dominating factors in orchestrating movements of the MTBD and MT interfacial segments in the dynein’s low-high affinity binding modes. Important interactions included β-Glu447 and β-Glu449 (CTT) with Arg3469 (MTBD-H6), Lys3472 (MTBD-H6-H7 loop) and Lys3479 (MTBD-H7); β-Glu449 with Lys3384 (MTBD-H8), Lys3386 and His3387 (MTBD-H1). The structural and precise position, orientation, and functional effects of the CTTs on the MT-MTBD, within reasonable cut-off distance for non-bonding interactions and under physiological conditions, are unavailable from the previous studies. The absence of the residues in the highly flexible MT-CTTs in the experimentally solved structures is perhaps in some cases due to insufficient data from density maps, but these segments are crucial in protein binding. The presented work contributes to the information useful for the MT-MTBD structure refinement

Large amplitude electromagnetic solitons in a fully relativistic magnetized electron-positron-pair plasma

11 pages, 7 figuresInternational audienceNonlinear propagation of purely stationary large amplitude electromagnetic (EM) solitary waves in a magnetized electron-positron (EP) plasma is studied using a fully relativistic two-fluid hydrodynamic model which accounts for physical regimes of both weakly relativistic $(P\ll nmc^2)$ and ultrarelativistic $(P\gg nmc^2)$ random thermal energies. Here, $P$ is the thermal pressure, $n$ the number density and $m$ the mass of a particle, and $c$ is the speed of light in vacuum. Previous theory in the literature [Phys. Plasmas \textbf{11}, 3078 (2004)] is advanced and generalized by the relativistic thermal motion of both electrons and positrons. While both the sub-Alfv{\'e}nic and super-Alfv{\'e}nic solitons coexist in the weakly relativistic regime, the ultrarelativistic EP plasmas in contrast support only the sub-Alfv{\'e}nic solitons. Different limits of the Mach numbers and soliton amplitudes are also examined in these two physical regimes

A chain of abrin and F1G4-IT have unique destinations.

<p>Cells treated with either 6 nM abrin or 50 nM F1G4-IT, for different intervals were subjected to sub-cellular fractionation. The nuclear (N), cytosolic (C) and organellar (O) fractions of each sample were electrophoresed on a 12.5% polyacrylamide SDS gel under reducing conditions and subjected to immunoblot analysis. <b>A:</b> Cells treated with abrin immunoblotted with mAb D6F10 for the A chain; Rabbit antibodies to acetylated histone, H3 (17 kDa), GAPDH (37 kDa) and Calnexin (67 kDa) were used as controls for nuclear, cytosolic and organellar fractions respectively. <b>B:</b> Cells treated with F1G4-IT immunoblotted with mAb D6F10; MAb to Lamin-A (70 kDa) and rabbit antibodies to GAPDH and Calnexin were used as controls for nuclear, cytosolic and organellar fractions respectively.</p

Abrin Immunotoxin: Targeted Cytotoxicity and Intracellular Trafficking Pathway

<div><p>Background</p><p>Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the <i>Abrus precatorius</i> plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy.</p> <p>Methods</p><p>Protein synthesis assay using ³[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins.</p> <p>Results</p><p>We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells.</p> <p>Conclusions</p><p>This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.</p> </div

Trigger of cell death by abrin-a A chain is independent of inhibition of protein synthesis.

<p>HepG2 cells (1×10⁶/ml) were treated with 19.2 nM of either one of the immunoconjugates: F1G4-IT or F1G4-IT<b>_R167L</b>, or abrin (51.25 pM) for different time intervals. The cells were harvested, fixed with 70% ethanol at −20°C, stained with staining solution (20 µg/ml propidium iodide and 50 µg/ml RNase A in PBS) and analyzed by flow cytometry. The percentage of dead population was determined and plotted above control cells. Each bar represents the mean of at least three different experiments carried out with duplicate samples. **P<0.005.</p

TrxR inhibitor rescues cells from F1G4-IT activity.

<p>HepG2 cells (1×10⁶/ml) were treated with auranofin for 6 h and cultured in presence of IC₉₀ of either abrin (51.25 pM) or F1G4-IT (19.2 nM) for 6 h in RPMI minus leucine. The cells were then pulsed with ³[H] L-leucine for 2 h, total protein precipitated with 5% TCA, solubilized with 0.1 N NaOH containing 1% SDS and the incorporated radioactivity measured. The percent radioactivity above control was determined. The graph depicts the mean of at least three different experiments carried out with duplicate samples. **P<0.05.</p

Kinetics of inhibition of protein synthesis by F1G4-IT is slower than that of abrin.

<p>HepG2 cells (1×10⁶/ml) were treated with IC₉₀ of either abrin or F1G4-IT for different time intervals and the procedure followed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058304#pone-0058304-g001" target="_blank">Figure 1</a>. The Boltzmann curve was used to analyze the data. The graph represents the mean of three separate experiments carried out with duplicate samples.</p

Intracellular localization of F1G4-IT in HepG2 cells is different from that of abrin.

<p>Cells (5×10⁶) treated with either abrin or F1G4-IT for different time intervals were fixed with 4% para-formaldehyde and stained with mAb D6F10-Alexa 488 for 2 h in the dark at RT. The cells were counter stained with 5 µg/ml of Hoechst 33342 for 10 min at RT, washed with PBS, mounted on slides and images acquired in the Zeiss confocal scanning microscope. The images were analysed using the Image J image browser. Confocal microscopy of, <b>A:</b> abrin treated cells; <b>B:</b> F1G4-IT treated cells.</p

Novel intracellular trafficking of abrin and its IT.

<p>F1G4-IT binds to the GnRH receptor via the antibody, mAb F1G4, and internalized via receptor-mediated endocytosis through clathrin coated pits. The protein is then released from the vesicles into the cytosol where the S-S bond between rABRa-A and the cross-linker SMPT is cleaved by thioredoxin, releasing the recombinant A chain. The thioredoxin, on the other hand, gets oxidized which is reduced back by the enzyme, thioredoxin reductase, using protons donated by cytosolic NADPH. This pathway is different from that observed for abrin, shown in the right half of the figure, wherein the internalized protein follows the retrograde pathway to reach the ER. In the ER, the disulfide bond is cleaved, releasing the A chain to the cytosol through the ERAD pathway. Once in the cytosol, irrespective of the pathway followed, the A chain binds to the 60S ribosomal subunit, depurinating the 28S rRNA, thus inhibiting protein synthesis.</p