Transfection by cationic gemini lipids and surfactants.

Diseases that are linked to defective genes or mutations can in principle be cured by gene therapy, in which damaged or absent genes are either repaired or replaced by new DNA in the nucleus of the cell. Related to this, disorders associated with elevated protein expression levels can be treated by RNA interference via the delivery of siRNA to the cytoplasm of cells. Polynucleotides can be brought into cells by viruses, but this is not without risk for the patient. Alternatively, DNA and RNA can be delivered by transfection, i.e. by non-viral vector systems such as cationic surfactants, which are also referred to as cationic lipids. In this review, recent progress on cationic lipids as transfection vectors will be discussed, with special emphasis on geminis, surfactants with 2 head groups and 2 tails connected by a spacer.

NMR detection in biofluid extracts at sub-µM concentrations via para-H2 induced hyperpolarization.

NMR spectroscopy is one of the most powerful techniques to simultaneously obtain qualitative and quantitative information in chemical analysis. Despite its versatility, the applications of NMR in the study of biofluids are often limited by the insensitivity of the technique, further aggravated by the poor signal dispersion in the (1)H spectra. Recent advances in para-H2 induced hyperpolarization have proven to address both these limitations for specific classes of compounds. Herein, this approach is for the first time applied for quantitative determination in biofluid extracts. We demonstrate that a combination of solid phase extraction, para-hydrogen induced hyperpolarization and selective NMR detection quickly reveals a doping substance, nikethamide, at sub-µM concentrations in urine. We suggest that this method can be further optimized for the detection of different analytes in various biofluids, anticipating a wider application of hyperpolarized NMR in metabolomics and pharmacokinetics studies in the near future.

Comparative Fe and Zn K-edge X-ray absorption spectroscopic study of the ferroxidase centres of human H-chain ferritin and bacterioferritin from Desulfovibrio desulfuricans.

Iron uptake by the ubiquitous iron-storage protein ferritin involves the oxidation of two Fe(II) ions located at the highly conserved dinuclear "ferroxidase centre" in individual subunits. We have measured X-ray absorption spectra of four mutants (K86Q, K86Q/E27D, K86Q/E107D, and K86Q/E27D/E107D, involving variations of Glu to Asp on either or both sides of the dinuclear ferroxidase site) of recombinant human H-chain ferritin (rHuHF) in their complexes with reactive Fe(II) and redox-inactive Zn(II). The results for Fe-rHuHf are compared with those for recombinant Desulfovibrio desulfuricans bacterioferritin (DdBfr) in three states: oxidised, reduced, and oxidised/Chelex-treated. The X-ray absorption near-edge region of the spectrum allows the oxidation state of the iron ions to be assessed. Extended X-ray absorption fine structure simulations have yielded accurate geometric information that represents an important refinement of the crystal structure of DdBfr; most metal-ligand bonds are shortened and there is a decrease in ionic radius going from the Fe(II) to the Fe(III) state. The Chelex-treated sample is found to be partly mineralised, giving an indication of the state of iron in the cycled-oxidised (reduced, then oxidised) form of DdBfr, where the crystal structure shows the dinuclear site to be only half occupied. In the case of rHuHF the complexes with Zn(II) reveal a surprising similarity between the variants, indicating that the rHuHf dinuclear site is rigid. In spite of this, the rHuHf complexes with Fe(II) show a variation in reactivity that is reflected in the iron oxidation states and coordination geometries.

Changes in the perception of biological X-ray absorption spectroscopy between ICBIC 1 and ICBIC 10/BIOXAS 2001: how far have we travelled?

The proceedings of BIOXAS 2001 (Siena, Italy, 2001) and both current and planned activities in the field of biological X-ray absorption spectroscopy are discussed against the perspective of changes in the perception of the technique since ICBIC 1 (Florence, Italy, 1983).

Synthesis, aggregation, and binding behavior of synthetic amphiphilic receptors.

Amphiphilic bowl-shaped receptor molecules have been synthesized starting from diphenylglycoluril. Upon dispersion in water, these molecules self-assemble to form vesicles that bind neutral guests and alkali metal ions. In the case of bis(alkylester)-modified receptor compound 4, electron microscopy reveals that an increase in the size of the alkali metal ion (from Na(+) or K(+) to Rb(+) and to Cs(+)) leads to a change in the shape of the aggregates, viz. from vesicles to tubules. Monolayer experiments suggest that this behavior is due to a change in the conformation of this amphiphilic receptor. In water, molecules of 4 have an elongated conformation that changes to a sandwich-like one upon binding of alkali metal ions. Binding studies with vesicles from the bis-ammonium receptors 6 and 9 and the guest 4-(4-nitrophenylazo)resorcinol (Magneson) reveal that below the critical aggregation concentration (CAC) of the amphiphile 1:1 host-guest complexes are formed with high host-guest association constants. Above the CAC, a host-guest ratio of 2:1 was observed that indicates that only the cavities on the outside of the vesicle can be occupied. In the case of the naphthalene walled compound 8 changes in the vesicle structure are induced by the organic guest Magneson.

Synthesis and supramolecular characterization of a novel class of glycopyranosyl-containing amphiphiles.

A novel class of glycopeptidolipids is described, which potentially can be used as a novel antigen-delivery system. The compounds have been prepared by a combination of solid-supported and solution-based methods. The use of the orthogonally protected FmocLysDde derivative provided an opportunity to incorporate two different types lipids. It was found that the model compound 1 forms aggregates in aqueous media which can be described as rod or tubelike structures. The aggregates can be stabilized by topotactic photopolymerization. Studies on the structural analogues 2-5 revealed the effect of the carbohydrate, peptide, and lipid moiety on the aggregation properties. It is concluded that none of the structure elements can lay claim to be exclusively important for the formation of highly organized aggregates such as tubes, fibers, or helical ribbons from 1, but the presence of all of these structural elements afforded the most uniformly shaped extended structures.

Changes in the iron coordination sphere of Fe(II) lipoxygenase-1 from soybeans upon binding of linoleate or oleate.

Fe K-edge X-ray absorption spectra of the non-heme iron constituent of lipoxygenase-1 from soybeans were obtained. The spectrum of 2.5 mM Fe(II) lipoxygenase, mixed with 1.2 M linoleate in the absence of O2, was compared to the spectrum of the native (i.e. untreated) enzyme. In the lipoxygenase-linoleate complex, an edge shift to lower energy was observed. This indicated that the iron-ligand distances in this complex are slightly longer than those in the untreated enzyme species. The extended X-ray absorption fine structure spectrum of Fe(II) lipoxygenase, prepared by anaerobic reduction of 2.5 mM Fe(II) lipoxygenase with 1.2 M linoleate, was very similar to the spectrum of the anaerobic lipoxygenase-linoleate complex. We conclude that the conformational differences between the iron coordination spheres of native and cycled Fe(II) lipoxygenase must be ascribed to the presence of linoleate, and not to changes in the enzyme that occur only after one cycle of oxidation and reduction. Furthermore, spectra of 2.5 mM Fe(II) lipoxygenase mixed with 1.2 M oleate, either in the absence or in the presence of O2, were also identical to the spectrum of the Fe(II) lipoxygenase-linoleate complex. This finding is in agreement with our observation that oleate is a competitive inhibitor of the lipoxygenase reaction. Moreover, the similarity of the lipoxygenase-oleate complexes in the presence and absence of O2 excludes the possibility that O2 binding to the iron cofactor is induced upon binding of a fatty acid to lipoxygenase.

X-ray absorption spectroscopy of soybean lipoxygenase-1. Influence of lipid hydroperoxide activation and lyophilization on the structure of the non-heme iron active site.

X-ray absorption spectra at the Fe K-edge of the non-heme iron site in Fe(II) as well as Fe(III) soybean lipoxygenase-1, in frozen solution or lyophilized, are presented; the latter spectra were obtained by incubation of the Fe(II) enzyme with its product hydroperoxide. An edge shift of about 2-3 eV to higher energy occurs upon oxidation of the Fe(II) enzyme to the Fe(III) species, corresponding to the valence change. The extended X-ray absorption fine structure shows clear differences in active-site structure as a result of this conversion. Curve-fitting on the new data of the Fe(II) enzyme, using the EXCURV88 program, leads to a coordination sphere that is in agreement with the active-site structure proposed earlier (6 +/- 1 N/O ligands at 0.205-0.209 nm with a maximum variance of 0.009 nm, including 4 +/- 1 imidazole ligands) [Navaratnam, S., Feiters, M. C., Al-Hakim, M., Allen, J. C., Veldink, G. A. and Vliegenthart, J. F. G. (1988) Biochim. Biophys. Acta 956, 70-76], while for the Fe(III) enzyme a shortening in ligand distances occurs (6 +/- 1 N/O ligands at 0.200-0.203 nm with maximum variance of 0.008 nm) and one imidazole is replaced by an oxygen ligand of unknown origin. Lyophilization does not lead to any apparent differences in the iron coordination of either species and gives a much better signal/noise ratio, allowing analysis of a larger range of data.

EXAFS of the type-1 copper site of rusticyanin.

Extended X-ray absorption fine structure (EXAFS) spectra at the Cu K-edge have been recorded of the oxidized and reduced form at pH 3.5 of rusticyanin, the type-1 or 'blue'-copper protein from Thiobacillus ferrooxidans. The EXAFS of oxidized rusticyanin is well simulated with models assuming a ligand set of 2 N(His) and 1 S(Cys) at 1.99 and 2.16 A, respectively. Upon reduction, the average Cu-N ligand distance increases by approx. 0.08A. For both redox states studied, the fit by the simulation is significantly improved by including a contribution of an additional sulfur ligand at approx. 2.8 A. From comparison with structural data of other blue-copper proteins, it is concluded that the copper coordination environment is relatively rigid, which may be a clue to its high redox potential.

Zinc environment in sheep liver sorbitol dehydrogenase.

The extended X-ray absorption fine structure (EXAFS) associated with the zinc K-absorption edge has been recorded for sorbitol dehydrogenase. It is interpreted in terms of one cysteine sulfur among the ligands to the active site zinc atom. Simulations of the EXAFS based on the presence of two such sulfurs are less satisfactory, and comparison with the EXAFS of such systems points to the presence of only one sulfur ligand in sorbitol dehydrogenase. These results provide evidence that sorbitol dehydrogenase does not have the characteristic one water, one His, two Cys arrangement of ligands to the active site zinc found in the homologous alcohol dehydrogenases and are consistent with the one water, one His, one Cys, one Glu ligand arrangement of the proposed model of sorbitol dehydrogenase [Eklund, H., Horjales, E., Jörnvall, H., Brändén, C.-I., & Jeffery J. (1985) Biochemistry 24, 8005-8012]. Evidence for the correctness of the model is also evidence for validity of predictive techniques used in constructing the model, i.e., computer graphics fitting of the amino acid sequence to the crystallographically derived structure of a different but homologous protein.

Spectroscopic investigations of Panulirus interruptus hemocyanin in the crystalline state.

Single-crystal ultraviolet spectroscopy, X-ray absorption spectroscopy and EPR measurements have been used to examine the oxidation and oxygenation state of the dinuclear copper site of several types of hemocyanin crystals. The crystals contain Panulirus interruptus hemocyanin which forms hexameric molecules with a molecular mass of approximately 470 kDa. Three types of crystals have been investigated. Type-I monoclinic crystals, which have been used for the X-ray structure determination, contain virtually only deoxyhemocyanin. Type-II monoclinic crystals, which are less well ordered than the type-I crystals, contain a mixture of deoxy, oxy and met forms. Older crystals contain relatively more methemocyanin. A third, hexagonal, crystal form is also partially oxygenated, and, like the type-II monoclinic form, subject to gradual conversion to methemocyanin.

Iron environment in soybean lipoxygenase-1.

The iron coordination in native, Fe(II), lipoxygenase has been studied by Extended X-Ray Absorption Fine Structure (EXAFS). The ligands are 6 +/- 1 nitrogen and/or oxygen ligands at 2.05-2.09 A, with a maximum variance of 0.09 A. The number of imidazole ligands is estimated at 4 +/- 1 using multiple scattering simulations. The remaining ligands are proposed to be carboxylate oxygens.

The metal site of stellacyanin: EXAFS studies of the Cu(II), Cu(I), Ni(II) and Co(II) derivatives.

Extended X-ray absorption fine structure (EXAFS) studies of Cu(II) (oxidized), Cu(I) (reduced), Ni(II) and Co(II) stellacyanin from Rhus vernicifera are reported. For Cu(II) stellacyanin, the coordination by three close ligands, viz. 2 N and 1 S, with the presence of smaller shells pointing to imidazole coordination, indicates similarities with the coordination in other so-called type 1 or 'blue'-copper proteins. Upon reduction, slightly longer ligand distances and an additional sulphur ligand are found. Ni(II) and Co(II) stellacyanin resemble Cu(I) and Cu(II) stellacyanin, respectively, in ligand distances, but have a tendency for three rather than two N (or O) ligands in the first shell. The results are compared with the three-dimensional model derived from 1H-NMR relaxation measurements for Co(II) stellacyanin, and are consistent with the proposal that apart from the three close ligands found in all blue-copper proteins, a sulphur from a disulphide bridge and the amide oxygen from an asparagine residue come to within coordinating distance of the metal in stellacyanin.

The pH and redox-state dependence of the copper site in azurin from Pseudomonas aeruginosa as studied by EXAFS.

The EXAFS of the K-edge of copper in azurin from Pseudomonas aeruginosa has been measured in solutions of the oxidized and reduced protein, at both low and high pH. Model compounds of known molecular structure, exhibiting Cu-N and Cu-S bonds of varying length, were studied as well. The major shell of the high-pH oxidized azurin EXAFS contains contributions of two N(His) at 1.95 +/- 0.03 A, and one S(Cys) at 2.23 +/- 0.03 A. Some minor contributions from the carbon atoms of the histidine residues and the distal sulfur atom are observed in the 3-4 A region. Upon reduction a decrease is seen in amplitude of the main peak in the Fourier transform, due to a lengthening of one of the Cu-N(His) bonds (2.05 +/- 0.03 A), and a shortening of the other (1.89 +/- 0.03 A), both by approx. 0.1 A. Indications for a Cu-S(Met) bond are found in the reduced azurin data (2.70 +/- 0.05 A). However, in the oxidized protein, this bond could not be determined unambiguously, in line with results of a model compound featuring weak Cu-thioether coordination. The effect of pH is only slight for both the oxidized and the reduced protein, and no significant changes in bond lengths are found upon a change of pH from 4.1 to 9.1. The relevance of these findings for the interpretation of the existing data on the redox activity of the protein is discussed.