Influence of Varying Water Quality Parameters on the Acute Toxicity of Silver to the Freshwater Cladoceran, Ceriodaphnia dubia.

The acute toxicity of silver to Ceriodaphnia dubia was investigated in laboratory reconstituted waters as well as in natural waters and reconstituted waters with natural organic matter. The water quality characteristics of the laboratory reconstituted waters were systematically varied. The parameters that demonstrated an ability to mitigate the acute toxic effects of silver were chloride, sodium, organic carbon, and chromium reducible sulfide. Factors that did not have a consistent effect on the acute toxicity of silver to C. dubia, at least over the range of conditions tested, included hardness, alkalinity, and pH. The biotic ligand model was calibrated to the observed test results and found to be of use in quantifying the effect of changing water quality characteristics on silver bioavailability and toxicity. The model generally predicted silver toxicity within a factor of two and should be useful in modifying water quality criteria.

The effect of food on the acute toxicity of silver nitrate to four freshwater test species and acute-to-chronic ratios.

Acute silver toxicity studies were conducted with and without food for four common freshwater test species: Daphnia magna, Ceriodaphnia dubia, Pimephales promelas (fathead minnow-FHM), and Oncorhynchus mykiss (rainbow trout-RBT) in order to generate acute-to-chronic ratios (ACR). The studies were conducted similarly (i.e., static-renewal or flow-through) to chronic/early-life stage studies that were previously performed in this laboratory. The acute toxicity (EC/LC50 values) of silver without food ranged from 0.57 µg dissolved Ag/l for C.dubia to 9.15 µg dissolved Ag/l for RBT. The presence of food resulted in an increase in EC/LC50 values from 1.25× for RBT to 22.4× for C. dubia. Invertebrate food type was also shown to effect acute silver toxicity. Food did not affect EC/LC50s or ACRs as greatly in fish studies as in invertebrate studies. ACRs for both invertebrate species were <1.0 when using acute studies without food but were 1.22 and 1.33 when using acute studies with food. ACRs for FHMs ranged from 4.06 to 7.19, while RBT ACRs ranged from 28.6 to 35.8 depending on whether food was present in acute studies. The data generated from this research program should be useful in re-determining a final ACR for silver in freshwater as well as in risk assessments.

Validation study of the acute biotic ligand model for silver.

An important final step in development of an acute biotic ligand model for silver is to validate predictive capabilities of the biotic ligand model developed for fish and invertebrates. To accomplish this, eight natural waters, collected from across North America, were characterized with respect to ionic composition, pH, dissolved organic carbon, and sulfide. Tests were conducted with the cladoceran Ceriodaphnia dubia (48-h static) and the fish Pimephales promelas (96-h static renewal) to determine the concentrations causing lethality to 50% of the organisms (LC50s) for silver in each of these waters. Overall, the biotic ligand model adequately predicted silver toxicity to C. dubia; however, in some cases, predicted LC50 values exceeded measured values. The accuracy of the biotic ligand model predictions was less convincing for silver toxicity to P. promelas with pronounced problems in low-ionic strength waters. Another issue was the use of acclimated organisms in toxicity studies because the biotic ligand model has been developed with the use of a mix of studies with acclimated and nonacclimated test organisms of varying ages and sizes. To evaluate whether effects of acclimation to test waters influence biotic ligand model predictions, a subset of the natural waters were also tested with P. promelas that had been acclimated to the natural water for 7 d before testing. These experiments revealed no differences in toxicity between acclimated and nonacclimated P. promelas. To determine the influence of organism size, which has been previously correlated to Na(+) turnover and acute silver toxicity across multiple species, Na(+) and Cl(-) influx rates were measured in P. promelas of different sizes. Our results show that Na(+) and Cl(-) influx rates were inversely related to fish mass and positively correlated with silver sensitivity.

S-layer variation in Bacillus stearothermophilus PV72 is based on DNA rearrangements between the chromosome and the naturally occurring megaplasmids.

Bacillus stearothermophilus PV72 expresses different S-layer genes (sbsA and sbsB) under different growth conditions. No stretches of significant sequence identity between sbsA and sbsB were detected. In order to investigate S-layer gene regulation in B. stearothermophilus PV72, we characterized the upstream regulatory region of sbsA and sbsB by sequencing and primer extension analysis. Both genes are transcribed from unique but different promoters, independently of the growth phase. Localization of sbsB in the sbsA-expressing strain PV72/p6 revealed that the coding region of the second S-layer gene sbsB is located not on the chromosome but on a natural megaplasmid of the strain, whereas the upstream regulatory region of sbsB was exclusively detected on the chromosome of PV72/p6. For sbsB expression, the coding region has to be integrated into the chromosomally located expression site. After the switch to sbsB expression, the sbsA coding region was removed from the chromosome but could still be detected on the plasmid of the sbsB-expressing strain PV72/p2. The sbsA upstream regulatory region, however, remained on the chromosome. This is the first report of S-layer variation not caused by intrachromosomal DNA rearrangements, but where variant formation depends on recombinational events between the plasmid and the chromosome.

The transposable element IS4712 prevents S-layer gene (sbsA) expression in Bacillus stearothermophilus and also affects the synthesis of altered surface layer proteins.

Cell surface (S)-layer protein synthesis in Bacillus stearothermophilus PV72/p6 is blocked when cells are grown at elevated temperature. From a culture exhibiting the S-layer-negative phenotype, the S-layer deficient mutant T5 (SbsA-) was isolated. Genetic analysis of the S-layer-encoding gene (sbsA) of mutant T5 revealed an insertion element (IS4712) integrated into the upstream regulatory region of the S-layer gene, thereby blocking sbsA transcription. The insertion element consists of 1371 base pairs which are flanked by two perfect inverted terminal repeats. Sequence similarity to other transposases of the IS4 family was detected. DNA-DNA hybridizations demonstrated that multiple homologues of IS4712 were also present within the genomes of several other thermophilic bacillus isolates. Attempts to isolate SbsA+ revertants failed. Instead, cells with altered surface proteins were detected. The synthesis of the altered S-layer proteins was correlated with the presence of IS4712 along with the occurrence of deletions in the sbsA coding region. Furthermore imprecise excision of IS4712 was detected. This work demonstrated that B. stearothermophilus is able to express at least four different S-layer proteins and that blocking of sbsA transcription by the insertion element IS4712 is associated with the expression of altered surface proteins.

Surface-accessible residues in the monomeric and assembled forms of a bacterial surface layer protein.

The S-layer protein SbsB of the thermophilic, Gram-positive organism Bacillus stearothermophilus PV72/p2 forms a crystalline, porous array constituting the outermost component of the cell envelope. SbsB has a molecular mass of 98 kDa, and the corresponding S-layer exhibits an oblique lattice symmetry. To investigate the molecular structure and assembly of SbsB, we replaced 75 residues (mainly serine, threonine, and alanine), located throughout the primary sequence, with cysteine, which is not found in the wild-type protein. As determined by electron microscopy, 72 out of 75 mutants formed regularly-structured self-assembly products identical to wild-type, thereby proving that the replacement of most of the selected amino acids by cysteine does not dramatically alter the structure of the protein. The three defective mutants, which showed a greatly reduced ability to self-assemble, were, however, successfully incorporated into S-layers of wild-type protein. Monomeric SbsB mutants and SbsB mutants assembled into S-layers were subjected to a surface accessibility screen by targeted chemical modification with a 5-kDa hydrophilic cysteine-reactive polyethylene glycol conjugate. In the monomeric form of SbsB, 34 of the examined residues were not surface accessible, while 23 were classified as very accessible, and 18 were of intermediate surface accessibility. By contrast, in the assembled S-layers, 57 of the mutated residues were not accessible, six were very accessible, and 12 of intermediate accessibility. Together with other structural information, the results suggest a model for SbsB in which functional domains are segregated along the length of the polypeptide chain.

Two-unit cantilevered resin-bonded fixed partial dentures--a retrospective, preliminary clinical investigation.

PURPOSE: The aim of this study was to retrospectively evaluate the clinical retention and abutment movement of 2-unit cantilevered resin-bonded fixed partial dentures (FPD) that were inserted at Prince Philip Dental Hospital in Hong Kong. MATERIALS AND METHODS: Of 45 patients who were identified from a hospital computer search after receiving a 2-unit cantilevered resin-bonded FPD, 31 were clinically examined (33 FPDs). For each patient the following data were recorded: gender, age, cementation date, endodontic treatment if performed, bone support, tooth mobility, and FPD tipping or drifting. Data about any debonds with subsequent treatment and patient satisfaction on a 10-point scale were also recorded. RESULTS: The mean service life for the 33 prostheses was 30 +/- 18 months, with a range of 72 days to 67 months. One prosthesis debonded, resulting in a clinical retention rate of 97%. No rotation, drifting, or tipping was observed for any of the prostheses during the short period of this study. CONCLUSION: Two-unit cantilevered resin-bonded FPDs are successful in the short term, but further research is required to determine if they offer a viable alternative to fixed-fixed resin-bonded FPD designs.

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Self-assembly product formation of the Bacillus stearothermophilus PV72/p6 S-layer protein SbsA in the course of autolysis of Bacillus subtilis.

In order to achieve high level expression and to study the release of a protein capable of self-assembly, the gene encoding the crystalline cell surface (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis. To obtain high level expression, a tightly regulated, xylose-inducible, stably replicating multicopy-plasmid vector was constructed. After induction of expression, the S-layer protein made up about 15% of the total cellular protein content, which was comparable to the SbsA content of B. stearothermophilus PV72/p6 cells. During all growth stages, SbsA was poorly secreted to the ambient cellular environment by B. subtilis. Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 65% of the synthesised SbsA was retained in the peptidoglycan-containing layer, indicating that the rigid cell wall layer was a barrier for efficient SbsA secretion. Electron microscopic investigation revealed that SbsA release from the peptidoglycan-containing layer started in the late stationary growth phase at distinct sites at the cell surface leading to the formation of extracellular self-assembly products which did not adhere to the cell wall surface. In addition, intracellular sheet-like SbsA self-assembly products which followed the curvature of the cell became visible in partly lysed cells. Intracellularly formed self-assembly products remained intact even after complete lysis of the rigid cell envelope layer.

New strategies for combination vaccines based on the extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.