Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG(4) encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 mug mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn(297) was not significantly affected by nocodazole during transient production by this method. (C) 2004 Wiley Periodicals, Inc.

A straightforward wet-chemical route to the nanocomposites of general layered clays and metal sulfides

The nanocomposites of general layered clays and metal sulfides could be produced from reactions of the layered clay aqueous suspensions and water-soluble metal-thiourea complexes. The clay could be saponite, montmorillonite, hectorite and laponite, while the metal sulfide could be cobalt sulfide, nickel sulfide, zinc sulfide, cadmium sulfide, and lead sulfide. In the nanocomposites, the clay could be incorporated with the metal sulfide pillars and metal sulfide nanoparticles. (c) 2006 Elsevier B.V. All rights reserved.

Dimeric allylpalladium(II) complexes with pyrazolate bridges: Synthesis, characterization, structure and thermal behaviour

The synthesis, characterization and thermal behaviour of some new dimeric allylpalladium (II) complexes bridged by pyrazolate ligands are reported. The complexes [Pd(mu-3, 5-R'(2)pz)(eta(3)-CH2C(R)CH2)](2) [R = H; R'= CH(CH3)(2) (1a); R = H, R' = C(CH3)(3) (1b), R = H; R' = CF3 (1c); R = CH3, R' = CH(CH3)(2) (2a); R = CH3, R' = C(CH3)(3) (2b); and R = CH3, R' = CF3 (2c)] have been prepared by the room temperature reaction of [Pd(eta(3)-CH2C(R)CH2)(acac)](acac = acetylacetonate) with 3,5-disubstituted pyrazoles in acetonitrile solution. The complexes have been characterized by NMR (H-1, C-13{H-1}), FT-IR, and elemental analyses. The structure of a representative complex, viz. 2c, has been established by single-crystal X-ray diffraction. The dinuclear molecule features two formally square planar palladium centres which are bridged by two pyrazole ligands and the coordination of each metal centre is completed by allyl substituents. The molecule has non-crystallographic mirror symmetry. Thermogravimetric studies have been carried out to evaluate the thermal stability of these complexes. Most of the complexes thermally decompose in argon atmosphere to give nanocrystals of palladium, which have been characterized by XRD, SEM and TEM. However, complex 2c can be sublimed in vacuo at 2 mbar without decomposition. The equilibrium vapour pressure of 2c has been measured by the Knudsen effusion technique. The vapour pressure of the complex 2c could be expressed by the relation: In (p/Pa)(+/- 0.06) = -18047.3/T + 46.85. The enthalpy and entropy of vapourization are found to be 150.0 +/- 3 kJ mol(-1) and 389.5 +/- 8 J K-1 mol(-1), respectively. (c) 2005 Elsevier B.V. All rights reserved.

Dinuclear cyano-bridged Co-III-Fe-II complexes as precursors for molecular mixed-valence complexes of higher nuclearity

The preparation and characterization of a series of trinuclear mixed-valence cyano-bridged Co-III-Fe-II-Co-III compounds derived from known dinuclear [{LnCoIII(mu-NC)}Fe-II(CN)(5)](-) complexes (L-n = N-5 or N3S2 n-membered pendant amine macrocycle) are presented. All of the new trinuclear complexes were fully characterized spectroscopically (UV-vis, IR, and C-13 NMR). Complexes exhibiting a trans and cis arrangement of the Co-Fe-Co units around the [Fe(CN)(6)](4-) center are described (i.e., cis/trans-[{LnCoIII(mu-NC)}(2)Fe-II(CN)(4)](2+)), and some of their structures are determined by X-ray crystallography. Electrochemical experiments revealed an expected anodic shift of the Fe-III/II redox potential upon addition of a tripositively charged {(CoLn)-L-III} moiety. The Co-III/II redox potentials do not change greatly from the di- to the trinuclear complex, but rather behave in a fully independent and noncooperative way. In this respect, the energies and extinction coefficients of the MMCT bands agree with the formal existence of two mixed-valence Fe-II-CN-Co-III units per molecule. Solvatochromic experiments also indicated that the MMCT band of these compounds behaves as expected for a class II mixed-valence complex. Nevertheless, its extinction coefficient is dramatically increased upon increasing the solvent donor number.

Complexes of cytotoxic chelators from the dipyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

In an effort to better understand the antiproliferative effects of the tridentate hydrazone chelators di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and di-2-pyridyl ketone benzoyl hydrazone (HPKBH), we report the coordination chemistry of these ligands with the divalent metal ions, Mn, Co, Ni, Cu, and Zn. These complexes are compared with their Fe-II analogues which were reported previously. The crystal structures of Co(PKIH)(2), Ni(PKIH)(2), Cu(PKIH)(2), Mn(PKBH)(2), Ni(PKBH)(2), Cu(PKBH)(2), and Zn(PKBH)(2) are reported where similar bis-tridenate coordination modes of the ligands are defined. In pure DMF, all complexes except the Zn-II compounds exhibit metal-centered M-III/II (Mn, Fe, Co, Ni) or M-II/I (Cu) redox processes. All complexes show ligand-centered reductions at low potential. Electrochemistry in a mixed water/DMF solvent only elicited metal-centered responses from the Co and Fe complexes. Remarkably, all complexes show antiproliferative activity against the SK-N-MC neuroepithelioma cell line similar to (HPKIH) or significantly greater than that of the (HPKBH) ligand which suggests a mechanism that does not only involve the redox activity of these complexes. In fact, we suggest that the complexes act as lipophilic transport shuttles that allow entrance to the cell and enable the delivery of both the ligand and metal which act in concert to inhibit proliferation.

Electronic energy-transfer rate constants for geometrical isomers of a bichromophoric macrocyclic complex

The rate of electronic energy transfer (EET) between a naphthalene donor and an anthracene acceptor in [ZnL3]-(ClO4)(2) and [ZnL4](ClO4)(2) was determined by time-resolved fluorescence measurements, where L 3 and L 4 are the geometrical isomers of 6-[(anthracen-9-ylmethyl)amino]-trans-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-13-amine (L-2), substituted with either a naphthalen-1-ylmethyl or naphthalen-2-ylmethyl donor, respectively. The energy transfer rate constant, k(EET), was determined to be (0.92 +/- 0.02) x 10(9) s(-1) for the naphthalen-1-ylmethyl-substituted isomer, while that for the naphthalen-2-ylmethyl-substituted isomer is somewhat faster, with k(EET) = (1.31 +/- 0.01) x 10(9) s(-1). The solid-state structure of [(ZnLCl)-Cl-3]ClO4 has been determined, and using molecular modeling calculations, the likely distributions of solution conformations in CH3CN have been evaluated for both complexes. The calculated conformational distributions in the common trans-III N-based isomeric form gave Forster EET rate constants that account for the differences observed and are in excellent agreement with the experimental values. It is shown that the full range of conformers must be considered to accurately reproduce the observed EET kinetics.

Isomeric distribution and catalyzed isomerization of cobalt(III) complexes with pentadentate macrocyclic ligands. Importance of hydrogen bonding

We have investigated the isomeric distribution and rearrangement of complexes of the type [CoXLn](2+,3+) (where X = Cl-, OH-, H2O, and L-n represents a pentadentate 13-, 14-, and 15-membered tetraaza or diaza-dithia (N-4 or N2S2) macrocycle bearing a pendant primary amine). The preparative procedures for chloro complexes produced almost exclusively kinetically preferred cis isomers (where the pendant primary amine is cis to the chloro ligand) that can be separated by careful cation-exchange chromatography. For L-13 and L-14 the so-called cis-V isomer is isolated as the kinetic product, and for L-15 the cis-VI form (an N-based diastereomer) is the preferred, while for the L-14(S) complex both cis-V and trans-I forms are obtained. All these complexes rearrange to form stable trans isomers in which the pendent primary amine is trans to the monodentate aqua or hydroxo ligand, depending on pH and the workup procedure. In total 11 different complexes have been studied. From these, two different trans isomers of [CoCIL14S](2+) have been characterized crystallographically for the first time in addition to a new structure of cis-V-[CoCIL14S](2+); all were isolated as their chloride perchlorate salts. Two additional isomers have been identified and characterized by NMR as reaction intermediates. The remaining seven forms correspond to the complexes already known, produced in preparative procedures. The kinetic, thermal, and baric activation parameters for all the isomerization reactions have been determined and involve large activation enthalpies and positive volumes of activation. Activation entropies indicate a very important degree of hydrogen bonding in the reactivity of the complexes, confirmed by density functional theory studies on the stability of the different isomeric forms. The isomerization processes are not simple and even some unstable intermediates have been detected and characterized as part of the above-mentioned 11 forms of the complexes. A common reaction mechanism for the isomerization reactions has been proposed for all the complexes derived from the observed kinetic and solution behavior.

Surface-functionalized polymer nanoparticles for selective sequestering of heavy metals

Xanthate-mediated (reversible addition-fragmentation chain transfer) emulsion polymerization has been used to create novel polystyrene nanoparticles with functionalized surfaces (see Figure) for the selective sequestering of heavy metals from water below ppm levels. These nanoparticles show a high degree of selectivity for Hg-II over Co-II. This technology has potential for the selective remediation of heavy metals from the human blood system.

Synthesis and characterization of Pd(II) and Pt(II) complexes with triazolopyrimidine derivatives: The crystal structure of [Pd2L2Cl4] where L=5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine

The Pd(II) and Pt(II) complexes with triazolopyrimidine C-nucleosides L-1 (5,7-dimethyl-3-(2',3',5'-tri-O-benzoyl-beta-D-ribofuranosyl-s-triazolo)[4,3-a]pyrimidine), L-2 (5,7-dimethyl-3-beta-D-ribofuranosyl-s-triazolo [4,3-a]pyrimidine) and L-3 (5,7-dimethyl[1,5-a]-s-triazolopyrimidine), [Pd(en)(L-1)](NO3)(2), (Pd(bpy)(L-1)](NO3)(2), cis-Pd(L-3)(2)Cl-2, [Pd-2(L-3)(2)Cl-4]center dot H2O, cis-Pd(L-2)(2)Cl-2 and [Pt-3(L-1)(2)Cl-6] were synthesized and characterized by elemental analysis and NMR spectroscopy. The structure of the [Pd-2(L-3)(2)Cl-4]center dot H2O complex was established by Xray crystallography. The two L-3 ligands are found in a head to tail orientation, with a (PdPd)-Pd-... distance of 3.1254(17) angstrom.L-1 coordinates to Pd(II) through N8 and N1 forming polymeric structures. L-2 coordinates to Pd(II) through N8 in acidic solutions (0.1 M HCl) forming complexes of cis-geometry. The Pd(II) coordination to L-2 does not affect the sugar conformation probably due to the high stability of the C-C glycoside bond. (c) 2006 Elsevier B.V. All rights reserved.

Cobalt complexes of tripodal hexadentate ligands: Electrochemically driven rearrangements

The Co-III complexes of the hexadentate tripodal ligands HOsen (3-(2'-aminoethylamino)-2,2-bis((2 ''-aminoethylamino) methyl) propan-1-ol) and HOten (3-(2'-aminoethylthia)-2,2-bis((2 ''-aminoethylthia) methyl) propan-1-ol) have been synthesized and fully characterized. The crystal structures of [Co(HOsen)]Cl-3 center dot H2O and [Co(HOten)](ClO4)Cl-2 are reported and in both cases the ligands coordinate as tripodal hexadentate N-6 and N3S3 donors, respectively. Cyclic voltammetry of the N3S3 coordinated complex [Co(HOten)](3+) is complicated and electrode dependent. On a Pt working electrode an irreversible Co-III/II couple ( formal potential - 157 mV versus Ag-AgCl) is seen, which is indicative of dissociation of the divalent complex formed at the electrode. The free HOten released by the dissociation of [Co(HOten)](2+) can be recaptured by Hg as shown by cyclic voltammetry experiments on a static Hg drop electrode ( or in the presence of Hg2+ ions), which leads to the formation of an electroactive Hg-II complex of the N3S3 ligand (formal potential + 60 mV versus Ag-AgCl). This behaviour is in contrast to the facile and totally reversible voltammetry of the hexaamine complex [Co(HOsen)](3+) ( formal potential (Co-III/II) - 519 mV versus Ag-AgCl), which is uncomplicated by any coupled chemical reactions. Akinetic and thermodynamic analysis of the [Co(HOten)](2+)/[Hg(HOten)](2+) system is presented on the basis of digital simulation of the experimental voltammetric data.

Hydrogen generation from liquid phase catalytic reforming of sugar solutions using metal-supported catalysts

Most of the hydrogen production processes are designed for large-scale industrial uses and are not suitable for a compact hydrogen device to be used in systems like solid polymer fuel cells. Integrating the reaction step, the gas purification and the heat supply can lead to small-scale hydrogen production systems. The aim of this research is to study the influence of several reaction parameters on hydrogen production using liquid phase reforming of sugar solution over Pt, Pd, and Ni supported on nanostructured supports. It was found that the desired catalytic pathway for H-2 production involves cleavage of C-C, C-H and O-H bonds that adsorb on the catalyst surface. Thus a good catalyst for production of H2 by liquid-phase reforming must facilitate C-C bond cleavage and promote removal of adsorbed CO species by the water-gas shift reaction, but the catalyst must not facilitate C-O bond cleavage and hydrogenation of CO or CO2. Apart from studying various catalysts, a commercial Pt/gamma-alumina catalyst was used to study the effect of temperature at three different temperatures of 458, 473 and 493 K. Some of the spent catalysts were characterised using TGA, SEM and XRD to study coke deposition. The amorphous and organised form of coke was found on the surface of the catalyst. (C) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Generic Technique to Generate Large Branched DNA Complexes

The inherent self-recognition properties of DNA have led to its use as a scaffold for various nanotechnology self-assembly applications, with macromolecular complexes, metallic and semiconducting nanoparticles, proteins, inter alia, being assembled onto a designed DNA scaffold. Such structures may typically comprise a number of DNA molecules organized into macromolecules. Many studies have used synthetic methods to produce the constituent DNA molecules, but this typically constrains the molecules to be no longer than around 100 base pairs (30 nm). However, applications that require larger self-assembling DNA complexes, several tens of nanometers or more, need to be generated by other techniques. Here, we present a generic technique to generate large linear, branched, and/or circular DNA macromolecular complexes. The effectiveness of this technique is demonstrated here by the use of Lambda Bacteriophage DNA as a template to generate single- and double-branched DNA structures approximately 120 nm in size.

Nickel(II) complexes of whole set of the simple ethylenediaminetetracarboxylate ligands:DFT study of complexes invoking molecular orbital and configurational analysis

The whole set of the nickel(II) complexes with no derivatized edta-type hexadentate ligands has been investigated from their structural and electronic properties. Two more complexes have been prepared in order to complete the whole set: trans(O5)-[Ni(ED3AP)]2- and trans(O5O6)-[Ni(EDA3P)]2- complexes. trans(O5) geometry has been verified crystallographically and trans(O5O6) geometry of the second complex has been predicted by the DFT theory and spectral analysis. Mutual dependance has been established between: the number of the five-membered carboxylate rings, octahedral/tetrahedral deviation of metal-ligand/nitrogen-neighbour-atom angles and charge-transfer energies (CTE) calculated by the Morokuma’s energetic decomposition analysis; energy of the absorption bands and HOMO–LUMO gap.