[Nanoparticulate silver. A new antimicrobial substance for bone cement]. / Nanopartikuläres Silber. Ein neues antimikrobielles Agens für Knochenzement.

BACKGROUND: Multiresistant bacteria have become an important problem in prosthetic joint infections. Their frequent resistance against gentamicin, which is commonly used in antibiotic-loaded bone cements, makes a new prophylaxis necessary. METHODS: PMMA-cement was loaded with 1% nanoparticulate silver and its antibacterial activity tested in vitro against gentamicin-resistant MRSE and MRSA strains as well as being compared to the activity of plain and gentamicin-loaded bone cements. A quantitative elution testing was also done to study the potentially cytotoxic effects of NanoSilver cement. RESULTS: Unloaded and PMMA-cement loaded with 2% gentamicin did not exhibit any antibacterial activity against MRSE and MRSA. At 1%, NanoSilver cement completely inhibited the proliferation of MRSA and MRSE. NanoSilver bone cement did not show any significant differences compared to the non-toxic control group. CONCLUSIONS: If these promising in vitro results can be confirmed in vivo, NanoSilver bone cement may be of considerable value in total joint arthroplasty.

Design of helical proteins for real-time endoprotease assays.

Proteases play a key role in cellular biology and have become priority targets for new pharmaceuticals. Thus, there is a high demand for specific, sensitive, and quick assays to monitor the activity of endoproteases. We designed affinity-tagged helical proteins with unique protease cleavage sites and thus constructed universal, molecularly defined, and uniform substrates for in vitro detection of IgA endoprotease. The substrate is a 10.5-kDa recombinant helical protein with a high-affinity (His)(6)-tag at the amino-terminal end. Further elements are a unique proteolytic recognition site and a C-terminal helical extension, which is cut off by the protease. Proteolytic action can be monitored in real time using surface plasmon resonance spectroscopy. Femtomole amounts of protease could be reliably and quantitatively detected within a few minutes after the start of the reaction. The detection signal changed linearly with the amount of protease and was independent of the applied sample flow rate. The biochip can be reversibly loaded with the recombinant protease substrate, so that the SPR assay is well-suited for automation. By substituting an HIV protease site for the recognition site of the IgAse, we also obtained a substrate for the quantitative and sensitive detection of HIV-1 endoprotease. Our substrate design is thus generally applicable.

Enzymatic microtiter plate-based nitrate detection in environmental and medical analysis.

Our microtiter plate assay is based on the enzymatic reduction of nitrate by dissimilatory nitrate reductase from Pseudomonas stutzeri [EC 1.7.99.4]. Exogenous redox mediators like methyl viologen, methylene blue, and cibachron blue were applied to reduce nitrate reductase. Concentrations of 0.02-0.9 mM nitrate can be detected with +/-6% standard deviation, by using a photometric Griess reaction for the formed nitrite. Nitrate reductase is stable in the pH range 6.5-9.0 and works in the temperature range 4-76 degrees C. The assay shows no interferences with salt content up to 1 M chloride or 11 mM chlorate, and serum albumin content up to 50 mg/ml. The time demand of our two-step procedure is 20 min/100 samples. Nitrate reductase could be conserved on site of the wells of microtiter plates for at least 6 months at room temperature. The nitrate assay was applied in environmental and consumer goods analysis, and for medical diagnostics in human plasma samples.

Imaging the activity of nitrate reductase by means of a scanning electrochemical microscope.

Scanning electrochemical microscopy (SECM) was used to characterize immobilized nitrate reductase (NaR) from Pseudonomonas stutzeri (E.C. 1.7.99.4). Nitrate reductase with membrane fragment was embedded in a polyurethane hydrogel in a capillary and solubilized NaR without membrane fragment was covalently coupled to a diaminoethyl-cellulose-carbamitate film on glass. After systematic studies of possible mediators, SECM feedback imaging of both forms of immobilized NaR was accomplished with methylviologen as redox mediator.

Comparative resonance Raman study of cytochrome c oxidase from beef heart and Paracoccus denitrificans.

Well-resolved, Soret band excited resonance Raman spectra were measured from the fully oxidized and fully reduced cytochrome c oxidase from beef heart and Paracoccus denitrificans. The vibrational patterns in the marker band region (1450-1700 cm-1) were analyzed, and a complete assignment of heme a and heme a3 vibrational modes is presented, permitting a detailed structural comparison of the mammalian and bacterial enzymes. Similar frequencies of the porphyrin modes for the reduced heme a and the reduced and oxidized heme a3 are found, indicating a close relationship of the ground-state conformations in all oxidase species studied. In oxidized heme a, however, significant frequency differences are observed and interpreted in terms of a ruffled porphyrin structure in the three- and two-subunit forms of the Paracoccus enzyme compared to the planar heme a of beef heart oxidase. The structural distortions, which also perturb the conformation of the formyl substituent and its electronic coupling with the porphyrin, reflect the specific heme-protein interactions at heme a. Since in the fully reduced state heme a appears to be largely planar in all oxidase species, the redox-linked conformational transition requires a more drastic rearrangement of the heme a-protein interactions in the bacterial than in the mammalian oxidase. For both heme a and heme a3 in the reduced state and for heme a3 in the oxidize state, frequency, intensity, and bandwidth differences of the formyl stretching vibration and intensity differences of some porphyrin modes are noted between the three oxidase forms. The same modes are also affected by quaternary structure changes in the bovine oxidase caused by different detergents and isolation procedures. These effects are attributed to differences of the dielectric properties of the heme environment, due to subtle structural changes in the heme pockets, induced by protein-protein interactions of subunit III with subunits I and/or II.

Genetics of Paracoccus denitrificans.

In bioenergetic research Paracoccus denitrificans has been used as an interesting model to elucidate the mechanisms of bacterial energy transduction. Genes for protein complexes of the respiratory chain and for proteins which are involved in periplasmic electron transport have been cloned and sequenced. Conjugational gene transfer has allowed the construction of site-specific mutant strains. Complementation experiments did not only open the field for site-directed mutagenesis and investigation of the structure/function relationship of the various electron-transport proteins, but also allowed first insights into processes like oxygen-dependent gene regulation or the assembly of electron-transport complexes. Also data will be presented that characterize two restriction-/modification systems, the codon usage and the promoter sequences of Paracoccus. Details will be given about the extrachromosomal localization of a duplicated cytochrome oxidase subunit I gene on one of the Paracoccus megaplasmids.

Paracoccus denitrificans mutants deleted in the gene for subunit II of cytochrome c oxidase also lack subunit I.

As a prerequisite to site-directed mutagenesis on cytochrome c oxidase, two different mutants are constructed by inactivating the cta gene locus encoding subunits II and III (ctaC and ctaE) of the Paracoccus denitrificans oxidase. Either a short fragment encoding part of the putative copper binding site near the C terminus of subunit II, or a substantial fragment, comprising parts of the coding region for both subunits and all of the intervening three open reading frames, are removed and replaced by the kanamycin resistance gene. Each construct, ligated into a suicide vector, is mated into Paracoccus, and mutants originating from double homologous recombination events are selected. We observe complete loss of alpha-type heme and of oxidase subunits, as well as a substantial decrease in the cytochrome c oxidase activity. Upon complementation with the ctaC gene (plus various lengths of downstream sequence extending into the operon), subunit II gets expressed in all cases. Wild-type phenotype, however, is only restored with the whole operon. Using smaller fragments for complementation gives interesting clues on roles of the open reading frames for the assembly process of the oxidase complex; two of the open reading frame genes most likely code for two independent assembly factors. Since homologous genes have been described not only for other bacterial oxidases, but their gene products shown to participate also in the assembly of the yeast enzyme, they seem to constitute a group of evolutionary conserved proteins.

The cytochrome c reductase/oxidase respiratory pathway of Paracoccus denitrificans: genetic and functional studies.

Data are presented on three components of the quinol oxidation branch of the Paracoccus respiratory chain: cytochrome c reductase, cytochrome c552, and the a-type terminal oxidase. Deletion mutants in the bc1 and the aa3 complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochrome c552 in electron transport between complex III and IV is presented.

Paracoccus denitrificans cytochrome c1 gene replacement mutants.

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.

Subunit II of cytochrome c oxidase from Paracoccus denitrificans. DNA sequence, gene expression and the protein.

Cytochrome c oxidase from the bacterium Paracoccus denitrificans, while being related to the mitochondrial enzyme in many ways, consists of only two to three different subunits. For the identification of its genes, a Paracoccus DNA library was constructed and screened with specific antibodies for expression of cloned inserts in E. coli. A positive clone expressing immunoreactive products in the molecular mass region of authentic subunit II revealed a high homology of its DNA-deduced amino acid sequence with subunit II sequences of the mitochondrial oxidases; several typical features, such as the transmembrane folding pattern and the presumed copper-binding site, are highly conserved between prokaryotic and mitochondrial polypeptides. A comparison with peptide sequencing data of the purified subunit established the presence of a characteristic N-terminal extension as well as a longer C terminus in the initial translation product of the Paracoccus subunit; by mass spectroscopy, the first N-terminally blocked residue of the mature polypeptide was identified as a pyroglutamate. No code abnormalities, but a highly specific codon usage were observed; no evidence for a localization of the subunit I gene directly adjacent to this gene has been obtained.