Photosensitized damage to liposome membranes was studied by using different dye-leakage assays based on fluorescence dequenching of a series of dyes upon their release from liposomes. Irradiation of liposomes with red light in the presence of a photosensitizer, trisulfonated aluminum phthalocyanine (AlPcS(3)), resulted in the pronounced leakage of carboxyfluorescein, but rather weak leakage of sulforhodamine B and almost negligible leakage of calcein from the corresponding dye-loaded liposomes. The same series of selectivity of liposome leakage was obtained with chlorin e6 that appeared to be more potent than AlPcS(3) in bringing about the photosensitized liposome leakage. Electrically neutral zinc phthalocyanine tetrasubstituted with a glycerol moiety (ZnPcGlyc(4)) was less effective than negatively charged AlPcS(3) in provoking the light-induced liposome permeabilization. On the contrary, both ZnPcGlyc(4) and AlPcS(3) were much more effective than chlorin e6 in sensitizing gramicidin channel inactivation in planar bilayer lipid membranes, thus showing that relative photodynamic efficacy of sensitizers can differ substantially for damaging different membrane targets. The photosensitized liposome permeabilization was apparently associated with oxidation of lipid double bonds by singlet oxygen as evidenced by the mandatory presence of unsaturated lipids in the membrane composition for the photosensitized liposome leakage to occur and the sensitivity of the latter to sodium azide. The fluorescence correlation spectroscopy measurements revealed marked permeability of photodynamically induced pores in liposome membranes for such photosensitizer as AlPcS(3).

The X-ray structure of mistletoe lectin I (MLI), a type-II ribosome-inactivating protein (RIP), cocrystallized with galactose is described. The model was refined at 3.0 Å resolution to an R-factor of 19.9% using 21 899 reflections, with R free 24.0%. MLI forms a homodimer (A-B) 2 in the crystal, as it does in solution at high concentration. The dimer is formed through contacts between the N-terminal domains of two B-chains involving weak polar and nonpolar interactions. Consequently, the overall arrangement of sugar-binding sites in MLI differs from those in monomeric type-II RIPs: two N-terminal sugar-binding sites are 15 Å apart on one side of the dimer, and two C-terminal sugarbinding sites are 87 Å apart on the other side. Galactose binding is achieved by common hydrogen bonds for the two binding sites via hydroxy groups 3-OH and 4-OH and hydrophobic contact by an aromatic ring. In addition, at the N-terminal site 2-OH forms hydrogen bonds with Asp27 and Lys41, and at the C-terminal site 3-OH and 6-OH undergo water-mediated interactions and C5 has a hydrophobic contact. MLI is a galactose-specific lectin and shows little affinity for N-acetylgalactosamine. The reason for this is discussed. Structural differences among the RIPs investigated in this study (their quaternary structures, location of sugar-binding sites, and fine sugar specificities of their B-chains, which could have diverged through evolution from a two-domain protein) may affect the binding sites, and consequently the cellular transport processes and biological responses of these toxins.

The goal of this study was to generate porous scaffolds from the genetically engineered protein, an analogue of Nephila clavipes spidroin 1 (rS1/9) and to assess the properties of new rS1/9 scaffolds essential for bioengineering. The salt leaching technique was used to make the rS1/9 scaffolds of interconnected macroporous structure with spontaneously formed micropores. The tensile strength of scaffolds was 18 ± 5 N/cm(2). Scaffolds were relatively stable in a phosphate buffer but degraded in oxidizing environment after 11 weeks of incubation. Applicability of the recombinant spidroin 1 as a substrate for cell culture was demonstrated by successful 3T3 cells growth on the surface of rS1/9 films (270 ± 20 cells/mm(2) vs. 97 ± 8 cells/mm(2) on the glass surface, p < 0.01). The 3T3 fibroblasts readily proliferated within the rS1/9 scaffold (from initially plated 19 ± 2 cells/mm(3) to 3800 ± 304 cells/mm(3) after 2 weeks). By this time, cells were uniformly distributed between the surface and deeper layers (27% ± 8% and 33% ± 4%, respectively; p > 0.05), whereas the initial distribution was 58% ± 7% and 11% ± 8%, respectively; p < 0.05). The rS1/9 scaffolds implanted subcutaneously into Balb/c mice were well tolerated. Over a 2-month period, the scaffolds promoted an ingrowth of de novo formed vascularized connective tissue elements and nerve fibers. Thus, scaffolds made of the novel recombinant spidroin 1 analogue are potentially applicable in tissue engineering.

The quaternary structure of mistletoe lectin I (MLI), a type II ribosome inactivating protein, has been determined by X-ray crystallography. A definitive molecular replacement solution was determined for MLI using the co-ordinates of the homologue ricin as a search model. MLI exists as an [AB] P dimer with internal crystallographic two-fold symmetry. Domain I of the B chains is non-covalently associated through interactions involving three looped chains (K K, L L, Q Q) in each molecule of the dimer, forming a double trefoil structure. The ricin molecule which shares 52% sequence homology with MLI has a disulphide bridge between Cys PH and Cys QW in the K K loop. An evolutionary mutation has replaced Cys QW with serine in MLI. This mutation appears to allow the K K loop the flexibility required to take up its place at the dimer interface, and also suggests a rationale for why ricin does not form dimers. Measurement of retention times using FPLC gel filtration confirms that dimerisation also occurs in solution between MLI B chains with an association constant K = 10 T M. z 1998 Federation of European Biochemical Societies.

No abstract

The quaternary structure of ricin agglutinin (RCA) has been determined by x-ray crystallography. The refined structure of ricin proved to be a successful search model using the molecular replacement method of phase determination. RCA forms an elongated molecule of dimensions 120 A x 60 A x 40 A with two A chains at the center and a B chain at each end. The A chains are covalently associated via a disulfide bridge between Cys 156 of both chains. Additional contacts at residues 114-5 stabilize the dimer interface. The covalent association of RCAA chains was confirmed by gel filtration under reducing and nonreducing conditions.

The layer-by-layer (LbL) deposition approach allows combined incorporation of fluorescent, magnetic, and plasmonic nanoparticles into the shell of polyelectrolyte microcapsules to obtain stimulus-responsive systems whose imaging and drug release functions can be triggered by external stimuli. The combined use of fluorescent quantum dots (QDs) and magnetic nanoparticles (MNPs) yields magnetic-field-driven imaging tools that can be tracked and imaged even deep in tissue when the appropriate type of QDs and wavelength of their excitation are used. QDs are excellent photonic labels for microcapsule encoding due to their close-to-unity photoluminescence (PL) quantum yields, narrow PL emission bands, and tremendous one-and two-photon extinction coefficients. However, the presence of MNPs and electrically charged polyelectrolyte molecules used for the LbL fabrication of magneto-optical microcapsules provokes alterations of the QD optical properties because of the photoinduced charge and energy transfer resulting in QD photodarkening or photobrightening. These lead to variation of the microcapsule PL signal under illumination, which hampers their tracking and quantitative analysis in cells and tissues. Here, we have studied the effects of the structure and spatial arrangement of the nanoparticles within the microcapsule polyelectrolyte shell, the total shell thickness, and the shell surface charge on their PL properties under continuous illumination. The roles of the charge transfer and its main driving forces in the stability of the microcapsules PL signal have been established, and the design of the microcapsules dually encoded with QDs and MNPs providing the strongest and most stable PL has been determined. Controlling the energy transfer from the QDs and MNPs and the charge transfer from QDs to polyelectrolyte layers in the engineering of magneto-optical microcapsules with a bright and stable PL signal extends their applications to long-lasting quantitative fluorescence imaging.

Quantum dot (QD) encoded microbeads are emerging for multiplexed analysis of biological markers. The quantitative encoding of microbeads prepared with different concentrations of QDs of different colors suffers from resonance energy transfer from the QDs fluorescing at shorter wavelengths to the QDs fluorescing at longer wavelengths. Here, we used the layer-by-layer deposition technique to spatially separate QDs of different colors with several polymer layers so that the distance between them would be larger than the Förster energy transfer radius. We performed fluorescence lifetime measurements to investigate and determine the conditions excluding significant resonance energy transfer between QDs within QD-encoded microbeads. Additionally, the number of QDs adsorbed onto microbeads was systematically established and multilayer structures of the QD-encoded microbead shells were characterized by scanning probe nanotomography. Finally, we prepared eight populations of FRET-free microbeads encoded with QDs of three colors at two intensity levels and demonstrated that all the optical codes are excitable at a single wavelength and may be clearly identified in three channels of a flow cytometer. The developed approach for engineering QD-encoded microbeads that are free from optical artefacts related to inter-QD resonance energy transfer paves the way to quantitative QD-based multiplexed assays.