Nanosilver-loaded PMMA bone cement doped with different bioactive glasses - evaluation of cytocompatibility, antibacterial activity, and mechanical properties.

Nanosilver-loaded PMMA bone cement (BC-AgNp) is a novel cement developed as a replacement for conventional cements. Despite its favorable properties and antibacterial activity, BC-AgNp still lacks biodegradability and bioactivity. Hence, we investigated doping with bioactive glasses (BGs) to create a new bioactive BC characterized by time-varying porosity and gradual release of AgNp. The BC Cemex was used as the base material and modified simultaneously with the AgNp and BGs: melted 45S5 and 13-93B3 glasses with various particle sizes and sol-gel derived SiO2/CaO microparticles. The effect of BG addition was examined by microscopic analysis, an assessment of setting parameters, wettability, FTIR and UV-VIS spectroscopy, mechanical testing, and hemo- and cytocompatibility and antibacterial efficiency studies. The results show that it is possible to incorporate various BGs into BC-AgNp, which leads to different properties depending on the type and size of BGs. The smaller particles of melted BGs showed higher porosity and better antibacterial properties with the moderate deterioration of mechanical properties. The sol-gel derived BGs, however, displayed a tendency for agglomeration and random distribution in BC-AgNp. The BGs with greater solubility more efficiently improve the antibacterial properties of BC-AgNp. Besides, the unreacted MMA monomer release could negatively influence the cellular response. Despite that, cements doped with different BGs are suitable for medical applications.

Design, characterization and in vitro evaluation of thin films enriched by tannic acid complexed by Fe(III) ions.

Materials based on carbohydrate polymers may be used for biomedical application. However, materials based on natural polymers have weak physicochemical properties. Thereby, there is a challenge to improve their properties without initiation of toxicity. The alternative method compared to toxic chemical agents' addition is the use of metal complexation method. In this study, chitosan/tannic acid mixtures modified by Fe(III) complexation are proposed and tested for potential applications as wound dressings. Thereby, surface properties, blood compatibility as well as platelet adhesion was tested. In addition, the periodontal ligament stromal cells compatibility studies were carried out. The results showed that the iron(III) addition to chitosan/tannic acid mixture improves properties due to a decrease in the surface free energy and exhibited a reduction in the hemolysis rate (below 5%). Moreover, cells cultured on the surface of films with Fe(III) showed higher metabolic activity. The current findings allow for the medical application of the proposed materials as wound dressings.

Influence of several biodegradable components added to pure and nanosilver-doped PMMA bone cements on its biological and mechanical properties.

Acrylic bone cements (BC) are wildly used in medicine. Despite favorable mechanical properties, processability and inject capability, BC lack bioactivity. To overcome this, we investigated the effects of selected biodegradable additives to create a partially-degradable BC and also we evaluated its combination with nanosilver (AgNp). We hypothesized that using above strategies it would be possible to obtain bioactive BC. The Cemex was used as the base material, modified at 2.5, 5 or 10 wt% with either cellulose, chitosan, magnesium, polydioxanone or tricalcium-phosphate. The resulted modified BC was examined for surface morphology, wettability, porosity, mechanical and nanomechanical properties and cytocompatibility. The composite BC doped with AgNp was also examined for its release and antibacterial properties. The results showed that it is possible to create modified cement and all studied modifiers increased its porosity. Applying the additives slightly decreased BC wettability and mechanical properties, but the positive effect of the additives was observed in nanomechanical research. The relatively poor cytocompatibility of modified BC was attributed to the unreacted monomer release, except for polydioxanone modification which increased cells viability. Furthermore, all additives facilitated AgNp release and increased BC antibacterial effectiveness. Our present studies suggest the optimal content of biodegradable component for BC is 5 wt%. At this content, an improvement in BC porosity is achieved without significant deterioration of BC physical and mechanical properties. Polydioxanone and cellulose seem to be the most promising additives that improve porosity and antibacterial properties of antibiotic or nanosilver-loaded BC. Partially-degradable BC may be a good strategy to improve their antibacterial effectiveness, but some caution is still required regarding their cytocompatibility. STATEMENT OF SIGNIFICANCE: The lack of bone cement bioactivity is the main limitation of its effectiveness in medicine. To overcome this, we have created composite cements with partially-degradable properties. We also modified these cements with nanosilver to provide antibacterial properties. We examined five various additives at three different contents to modify a selected bone cement. Our results broaden the knowledge about potential modifiers and properties of composite cements. We selected the optimal content and the most promising additives, and showed that the combination of these additives with nanosilver would increase cements` antibacterial effectiveness. Such modified cements may be a new solution for medical applications.

Physicochemical properties of scaffolds based on mixtures of chitosan, collagen and glycosaminoglycans with nano-hydroxyapatite addition.

Scaffolds based on chitosan (CTS), collagen (Coll), and glycosaminoglycans (GAGs) mixtures with nano-hydroxyapatite (HAp) were obtained with the use of the freeze-drying method. They were characterized by different analyses, e.g. SEM images and mechanical testing. Moreover, swelling behavior and biocompatibility tests were carried out. The results showed that the scaffolds based on the blends of chitosan, collagen, and glycosaminoglycans with hydroxyapatite are stable in aqueous environment. SEM images allowed the observation of a porous scaffolds structure with the pores size ~250 µm. The main purpose of the research was to detect the influence of hydroxyapatite addition on the glycosaminoglycans-enriched scaffolds properties. The physicochemical properties as swelling and mechanical parameters were tested. The scaffolds structure was observed by SEM. Moreover, the preliminary assessment of scaffolds suitability for cell growth, human osteosarcoma cell line SaOS-2 was used. The obtained results indicate that the addition of hydroxyapatite improves the mechanical parameters and cells biological response of the studied materials.

In vivo study on scaffolds based on chitosan, collagen, and hyaluronic acid with hydroxyapatite.

Scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with nano-hydroxyapatite were obtained with the use of the freeze-drying method. Composites swelling behavior was assessed by the liquid uptake test. The adhesion and proliferation of human osteosarcoma SaOS-2 cells on the scaffolds were examined in 4-day culture. The biocompatibility of the chosen scaffolds was further studied by in vivo implantation into subcutaneous tissue of rabbits. The results showed low stability of the scaffolds based on chitosan, collagen, and hyaluronic acid supplemented with hydroxyapatite. The addition of hydroxyapatite delayed the degradation process of the obtained scaffolds. The X-ray images of the tissues surrounding the scaffolds showed that both, the control scaffold without hydroxyapatite (HAp) and those with addition of 50% wt. HAp underwent degradation after 6 months. However, the scaffolds supplemented with 80% wt. HAp premained in the implanted place. The results showed satisfactory tissue response on the implanted scaffolds.

The application of chitosan/collagen/hyaluronic acid sponge cross-linked by dialdehyde starch addition as a matrix for calcium phosphate in situ precipitation.

Scaffolds based on chitosan, collagen and hyaluronic acid, cross-linked by dialdehyde starch were obtained through the freeze-drying method. The porous structures were used as matrixes for calcium phosphate in situ precipitation. Composites were characterized by different analyses, e.g. infrared spectroscopy, SEM images, porosity, density, and mechanical tests. Moreover, an examination involving the energy dispersive X-ray spectroscopic method was carried out for the calcium and phosphorus ratio determination. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells were examined on the obtained scaffolds. The results showed that the properties of the scaffolds based on chitosan, collagen, and hyaluronic acid can be modified by dialdehyde starch addition. The mechanical parameters (i.e. compressive modulus and maximum compressive force), porosity, and density of the material were improved. Calcium phosphate was deposited in the scaffolds at the Ca/P ratio ∼2. SEM images showed the homogeneous structure, with interconnected pores. The cross-linker addition and an inorganic compound precipitation improved the biocompatibility of the scaffolds. The obtained materials can provide the support required in tissue engineering and regenerative medicine.

New composite materials prepared by calcium phosphate precipitation in chitosan/collagen/hyaluronic acid sponge cross-linked by EDC/NHS.

Nowadays, fabrication of composite materials based on biopolymers is a rising field due to potential for bone repair and tissue engineering application. Blending of different biopolymers and incorporation of inorganic particles in the blend can lead to new materials with improved physicochemical properties and biocompatibility. In this work 3D porous structures called scaffolds based on chitosan, collagen and hyaluronic acid were obtained through the lyophilization process. Scaffolds were cross-linked by EDC/NHS. Infrared spectra for the materials were made, the percentage of swelling, scaffolds porosity and density, mechanical parameters, thermal stability were studied. Moreover, the scaffolds were used as matrixes for the calcium phosphate in situ precipitation. SEM images were taken and EDX analysis was carried out for calcium and phosphorous content determination in the scaffold. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells was examined on obtained scaffolds. The results showed that the properties of 3D composites cross-linked by EDC/NHS were altered after the addition of 1, 2 and 5% hyaluronic acid. Mechanical parameters, thermal stability and porosity of scaffolds were improved. Moreover, calcium and phosphorous were found in each kind of scaffold. SEM images showed that the precipitation was homogeneously carried in the whole volume of samples. Attachment of SaOS-2 cells to all modified materials was better compared to unmodified control and proliferation of these cells was markedly increased on scaffolds with precipitated calcium phosphate. Obtained materials can provide the support useful in tissue engineering and regenerative medicine.

Characterization of gelatin and chitosan scaffolds cross-linked by addition of dialdehyde starch.

In this study the influence of the addition of dialdehyde starch on the properties of scaffolds based on gelatin and chitosan obtained by the freeze-drying method was investigated. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells on the obtained scaffolds was examined. Chitosan and gelatin were mixed in different weight ratios (75/25, 50/50, 25/75) with 1, 2 and 5 wt% addition of dialdehyde starch. The obtained scaffolds were subjected to mechanical testing, infrared spectroscopy, swelling measurements, low-pressure porosimetry and zeta potential measurement. Internal material structures were observed by scanning electron microscopy. The results showed that the cross-linking process occurred after the addition of dialdehyde starch and resulted in increased mechanical strength, swelling properties, zeta potential and porosity of studied materials. The attachment of SaOS-2 cells to all modified materials was better compared to an unmodified control and the proliferation of these cells was markedly increased on modified scaffolds.

Pathways of Transmembrane Electron Transfer in Cytochrome bc Complexes: Dielectric Heterogeneity and Interheme Coulombic Interactions.

The intramembrane cytochrome bc1 complex of the photosynthetic bacterium Rhodobacter capsulatus and the cytochrome b6f complex, which functions in oxygenic photosynthesis, utilize two pairs of b-hemes in a symmetric dimer to accomplish proton-coupled electron transfer. The transmembrane electron transfer pathway in each complex was identified through the novel use of heme Soret band excitonic circular dichroism (CD) spectra, for which the responsible heme-heme interactions were determined from crystal structures. Kinetics of heme reduction and CD amplitude change were measured simultaneously. For bc1, in which the redox potentials of the transmembrane heme pair are separated by 160 mV, heme reduction occurs preferentially to the higher-potential intermonomer heme pair on the electronegative (n) side of the complex. This contrasts with the b6f complex, where the redox potential difference between transmembrane intramonomer p- and n-side hemes is substantially smaller and the n-p pair is preferentially reduced. Limits on the dielectric constant between intramonomer hemes were calculated from the interheme distance and the redox potential difference, ΔEm. The difference in preferred reduction pathway is a consequence of the larger ΔEm between n- and p-side hemes in bc1, which favors the reduction of n-side hemes and cannot be offset by decreased repulsive Coulombic interactions between intramonomer hemes.

Incorporation of sol-gel bioactive glass into PLGA improves mechanical properties and bioactivity of composite scaffolds and results in their osteoinductive properties.

In this study, 3D porous bioactive composite scaffolds were produced and evaluated for their physico-chemical and biological properties. Polymer poly-L-lactide-co-glycolide (PLGA) matrix scaffolds were modified with sol-gel-derived bioactive glasses (SBGs) of CaO-SiO2-P2O5 systems. We hypothesized that SBG incorporation into PLGA matrix would improve the chemical and biological activity of composite materials as well as their mechanical properties. We applied two bioactive glasses, designated as S2 or A2, differing in the content of SiO2 and CaO (i.e. 80 mol% SiO2, 16 mol% CaO for S2 and 40 mol% SiO2, 52 mol% CaO for A2). The composites were characterized for their porosity, bioactivity, microstructure and mechanical properties. The osteoinductive properties of these composites were evaluated in human bone marrow stromal cell (hBMSC) cultures grown in either standard growth medium or treated with recombinant human bone morphogenetic protein-2 (rhBMP-2) or dexamethasone (Dex). After incubation in simulated body fluid, calcium phosphate precipitates formed inside the pores of both A2-PLGA and S2-PLGA scaffolds. The compressive strength of the latter was increased slightly compared to PLGA. Both composites promoted superior hBMSC attachment to the material surface and stimulated the expression of several osteogenic markers in hBMSC compared to cells grown on unmodified PLGA. There were also marked differences in the response of hBMSC to composite scaffolds, depending on chemical compositions of the scaffolds and culture treatments. Compared to silica-rich S2-PLGA, hBMSC grown on calcium-rich A2-PLGA were overall less responsive to rhBMP-2 or Dex and the osteoinductive properties of these A2-PLGA scaffolds seemed partially dependent on their ability to induce BMP signaling in untreated hBMSC. Thus, beyond the ability of currently studied composites to enhance hBMSC osteogenesis, it may become possible to modulate the osteogenic response of hBMSC, depending on the chemistry of SBGs incorporated into polymer matrix.

Photocrosslinked ultrathin anionic polysaccharide supports for accelerated growth of human mesenchymal stem cells.

OBJECTIVES: Properties of cell culture supports obtained from ultrathin multilayer films containing anionic natural polysaccharides (PSacs) and a synthetic polycation were studied. MATERIALS AND METHODS: Supports were prepared via a layer-by-layer (LbL) self-assembly deposition method. Polymers used were: heparin (Hep), chondroitin sulphate (CS), hyaluronic acid (HA), and ι-carrageenan (Car) as polyanions, and diazoresin (DR) as a polycation. PSac layers were crosslinked with DR layers by irradiation with UV light absorbed by DR resin. RESULTS: DR/PSac films are very efficient cell culture growth supports as found from experiments with human mesenchymal stem cells (hMSCs). Irradiation of the films resulted in changing zeta potential of outermost layers of both DR and PSac to more negative values, and in increased film hydrophobicity, as found from the contact angle measurements. Photocrosslinking of the supports led to their increased stability. CONCLUSIONS: The supports allow for obtaining intact cell monolayers faster than when typical polystyrene tissue culture plates are used. Moreover, these monolayers spontaneously detach permitting formation of new cell layers on these surfaces relatively early during culture, compared to cells cultured on commonly used tissue culture plastic.

Multilineage differentiation of adult human bone marrow progenitor cells transduced with human papilloma virus type 16 E6/E7 genes.

We have established a new adult human bone marrow-derived cell line hMPC 32F, stably transduced with human papilloma virus type 16 E6/E7 genes, that displays mesenchymal multilineage differentiation ability in vitro. The hMPC 32F cells exhibited a population doubling time of 22 h and have been maintained in culture for about 20 passages. When cultured in conditions promoting osteogenic, adipogenic, or chondrogenic differentiation, hMPC 32F cells expressed mature differentiated phenotypes. These include (1) osteoblastic phenotype characterized by upregulated alkaline phosphatase (ALP) expression and extracellular matrix mineralization, (2) adipocytic phenotype with the presence of intracellular lipid droplets, and (3) chondrocytic phenotype of round cells surrounded by a sulfated proteoglycan-rich matrix. In addition, the hMPC 32F cells expressed differentiation lineage-specific genes, as detected by RT-PCR. Furthermore, osteogenic and adipogenic cultures responded to regulatory factors such as transforming growth factor-beta1 (TGF-beta1) and 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3). Thus, continuous treatment of osteogenic cultures for 2 weeks with TGF-beta1 decreased ALP activity and mRNA expression and inhibited osteocalcin mRNA expression and matrix mineralization, whereas l,25(OH)2D3 had an additive, stimulatory effect. In adipogenic cultures, treatment with TGF-beta1 for 2 weeks markedly inhibited adipogenesis whereas 1,25(OH)2D3 had no obvious effect. Finally, clonal analysis of hMPC 32F cells revealed a high percentage of multipotent clones, although clones of more restricted differentiation potential were also present. These characteristics of the hMPC 32F cell line suggest their pluripotent, progenitor, and nontransformed nature and indicate their potential application for studying the mechanisms governing developmental potential of adult human bone marrow mesenchymal progenitor cells.

Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine.

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167. Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c. Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1). The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.

EPR studies of iso-1-cytochrome c: effect of temperature on two-component spectra of spin label attached to cysteine at positions 102 and 47.

Wild-type iso-1-cytochrome c from Saccharomyces cerevisiae containing naturally occurring cysteine at position 102 and mutated protein S47C (derived from the protein in which C102 had been replaced by threonine) were labeled with cysteine-specific methanethiosulfonate spin label. Continuous wave (CW) electron paramagnetic resonance (EPR) was used to examine the effect of temperature on the behavior of the spin label in the oxidized and reduced forms of wild-type cytochrome c and in the oxidized form of the mutated protein. The computer simulations revealed that the CW EPR spectrum for each form of cytochrome c consists of at least two components [a fast (F) and a slow (S) component], which differ in the values of the rotational correlation times tauRparallel (longitudinal rotational correlation time) and tauRperpendicular (transverse rotational correlation time) and that the relative contributions of the F and S components of the spectra change with temperature. In addition, the values of the rotational correlation times (tauRparallel and tauRperpendicular) for the F component appear to change much more dramatically with the temperature than the respective values for the S component. A large difference between the behavior of the oxidized and reduced wild-type spin-labeled cytochromes c indicates that the temperature-induced unfolding of the protein in the region around C102 progresses more rapidly when cytochrome c is in the oxidized form.

Different mechanisms of the binding of soluble electron donors to the photosynthetic reaction center of Rubrivivax gelatinosus and Blastochloris viridis.

The tetraheme cytochrome subunits of the photosynthetic reaction centers (RCs) in two species of purple bacteria, Rubrivivax gelatinosus and Blastochloris (Rhodopseudomonas) viridis, were compared in terms of their capabilities to bind different electron-donor proteins. The wild-type RCs from both species and mutated forms of R. gelatinosus RCs (with amino acid substitutions introduced to the binding domain for electron-donor proteins) were tested for their reactivity with soluble cytochromes and high potential iron-sulfur protein. Cytochromes from both species were good electron donors to the B. viridis RC and the R. gelatinosus RC. The reactivity in the R. gelatinosus RC showed a clear dependence on the polarity of the charges introduced to the binding domain, indicating the importance of the electrostatic interactions. In contrast, high potential iron-sulfur protein, presumed to operate according to the hydrophobic mechanism of binding, reacted significantly only with the R. gelatinosus RC. Evolutionary substitution of amino acids in a region of the binding domain on the cytochrome subunit surface probably caused the change in the principal mode of protein-protein interactions in the electron-transfer chains.

Gel-derived materials of a CaO-P(2)O(5)-SiO(2) system modified by boron, sodium, magnesium, aluminum, and fluorine compounds.

Bioactive glass-ceramic materials of the CaO-P(2)O(5)-SiO(2) system modified by adding boron, magnesium, sodium, fluorine, and aluminum were obtained using the sol-gel method. Gel-derived materials were produced in the pellet form obtained by compression of powders as well as in coatings on glass slides. The materials obtained were examined in vitro with regard to the ability of calcium phosphate layer to form on the material surface as the result of contact with simulated body fluid (SBF). SBF pH changes and calcium solubility in this solution were determined and scanning electron microscopy, energy-dispersive X-ray analysis, and infrared spectroscopy studies were conducted before and after contact of the materials with SBF. The gels modified by aluminum were amorphous, whereas the sodium and fluorine additives promoted the bulk crystallization of gel-derived materials. The ability of calcium phosphates to crystallize on the surface of gel-derived materials depended only slightly on the types of additives applied, and the character of this dependence was different from that observed in melted glasses. Moreover, to estimate the biocompatibility of gel-derived coatings, we examined the proliferation, collagen synthesis, adhesion, and morphology of fibroblasts (NRK cells) cultured in the presence of gel-derived materials. The results of these experiments showed that none of the tested materials significantly reduced any cell function.

Interaction site for high-potential iron-sulfur protein on the tetraheme cytochrome subunit bound to the photosynthetic reaction center of Rubrivivax gelatinosus.

We have recently demonstrated, using site-directed mutagenesis, that soluble cytochromes interact with the Rubrivivax gelatinosus photosynthetic reaction center (RC) in the vicinity of the low-potential heme 1 (c-551, Em = 70 mV) of the tetraheme cytochrome subunit, the fourth heme from the special pair of bacteriochlorophyll [Osyczka, A., et al. (1998) Biochemistry 37, 11732-11744]. Although the mutations generated in that study did not show clear effects on the electron transfer from high-potential iron-sulfur protein (HiPIP), which is the major physiological electron donor to the RC in this bacterium, we report here that other site-directed mutations near the solvent-exposed edge of the same low-potential heme 1, V67K (valine-67 substituted by lysine) and E79K/E85K/E93K (glutamates-79, -85, and -93, all replaced by lysines), considerably inhibit the electron transfer from HiPIP to the RC. Thus, it is concluded that HiPIP, like soluble cytochromes, binds to the RC in the vicinity of the exposed part of the low-potential heme 1 of the cytochrome subunit, although some differences in the configurations of the HiPIP-RC and cytochrome c-RC transient complexes may be postulated.

Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.

Comparison of the binding sites for high-potential iron-sulfur protein and cytochrome c on the tetraheme cytochrome subunit bound to the bacterial photosynthetic reaction center.

A tetraheme cytochrome subunit bound to the photosynthetic reaction center (RC) of purple bacterium, Rubrivivax gelatinosus, interacts with two types of soluble electron donors, cytochromes c and high-potential iron-sulfur protein (HiPIP), at a binding domain in the vicinity of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll. To clarify the mechanism of the interaction, the domain around heme 1 was examined using site-directed mutants that changed the surface charge in the region within 20 A from the heme edge. In the case of the interaction with soluble cytochrome c, a strong dependence on the sign of the introduced charge was observed in all mutants: positive charge inhibited the reaction rate, whereas additional negative charge accelerated it. This confirmed the electrostatic nature of the binding. Interaction with HiPIP was inhibited by a limited number of mutations at the close vicinity of heme 1, and no acceleration was observed (the effects of some mutations were independent of the sign of the introduced charge). The acidic residues which were critically important for the binding of cytochrome c showed much less contribution to the binding of HiPIP. The binding site for HiPIP appears to be mostly formed by uncharged and hydrophobic residues, occupying a significantly smaller area than the cytochrome-c-binding site. It is proposed that the docking of HiPIP to the RC in Rvi. gelatinosus is primarily controlled by hydrophobic contacts between protein surfaces, thus differing from the electrostatic mode of the RC-cytochrome c interaction.

Optimum design of a grazing-incidence flat-field spectrograph with a spherical varied-line-space grating.

A grazing-incidence spectrograph is designed by use of the flat-field image-focusing property of a spherical varied-line-space grating. Optimum grating parameters for mechanical ruling are selected by application of genetic algorithms. Two gratings, one for 2-5-nm and the other for 5-20-nm spectral regions, are designed, and their fabrication tolerances are analyzed.