Antimicrobial activity of silver nitrate against periodontal pathogens.

Metal ions were evaluated as potential antimicrobial agents suitable for local delivery in the oral cavity for the treatment of periodontitis. Silver nitrate, copper chloride, and zinc chloride were tested for antimicrobial activity in in vitro killing assays conducted in phosphate buffered saline with a series of oral bacteria including gram-negative periodontal pathogens and gram-positive streptococci. Copper and zinc salts failed to exhibit strong and consistent activity against periodontal pathogens. In contrast, silver at a concentration of 0.5 microg/mL produced a 3 log10 reduction in colony forming units (CFU)/mL or greater against all periodontal pathogens tested including Porphyromonas gingivalis, Prevotella intermedia, Prevotella denticola, Bacteroides forsythus, Fusobacterium nucleatum vincentii, Campylobacter gracilis, Campylobacter rectus, Eikenella corrodens, and Actinobacillus actinomycetemcomitans. In comparison, substantially higher concentrations of silver nitrate failed to kill oral streptococci. A silver nitrate concentration of 25 microg/mL produced log10 reductions in CFU/mL of 3.5-5 in killing assays performed in human serum against P. gingivalis, demonstrating the ability of silver to retain activity in a biological medium similar to that encountered in vivo in the periodontal pocket. These results identify silver nitrate, an antimicrobial that may possess advantages over traditional antibiotics, as a potential agent for controlled release local delivery in the oral cavity for the treatment of periodontitis.

Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5.

Through the analysis of a series of 25 peptides composed of various portions of the histatin 5 sequence, we have identified P-113, a 12-amino-acid fragment of histatin 5, as the smallest fragment that retains anticandidal activity comparable to that of the parent compound. Amidation of the P-113 C terminus increased the anticandidal activity of P-113 approximately twofold. The three histidine residues could be exchanged for three hydrophobic residues, with the fragment retaining anticandidal activity. However, the change of two or more of the five basic (lysine and arginine) residues to uncharged residues resulted in a substantial loss of anticandidal activity. A synthetic D-amino-acid analogue, P-113D, was as active against Candida albicans as the L-amino-acid form. In vitro MIC tests in low-ionic-strength medium showed that P-113 has potent activity against Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. These results identify P-113 as a potential antimicrobial agent in the treatment of oral candidiasis.

Photoactive porphyrin derivative with broad-spectrum activity against oral pathogens In vitro.

Photodynamic therapy (PDT) has historically been used as a means to treat cancerous tumors but has recently been used to kill bacterial cells through the use of targeted photosensitizers. PDT is a potential adjunct to scaling and root planing in the treatment of periodontal disease. However, the effectiveness of porphyrin derivatives against microorganisms has been limited because some gram-negative bacteria are refractory to photodynamic treatment with these agents. We have designed a porphyrin derivative conjugated to a pentalysine moeity that endows the molecule with activity against gram-positive and gram-negative bacteria. Whereas the porphyrin, chlorin e6, showed in vitro activity against a limited spectrum of bacteria, chlorin e6 conjugated to pentalysine showed in vitro activity against all oral microorganisms tested, including Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. polymorphum, Actinomyces viscosus, and the streptococci. Potent antimicrobial activity (>/=5-log-unit reduction in the numbers of CFU per milliliter) was retained in the presence of up to 25% whole sheep blood. The use of potent, selective agents such as this chlorin e6-pentalysine conjugate to more effectively reduce the pathogenic bacteria in the periodontal pocket may be a significant tool for the treatment of periodontal disease.

Sustained release of silver from periodontal wafers for treatment of periodontitis.

Periodontal wafers intended to treat the underlying infections in patients with periodontitis have been developed. The wafers consist of poly(lactic-co-glycolic acid) as a primary bioerodible polymeric component, poly(ethylene glycol) as a plasticizer and encapsulation aid, and silver nitrate as the antimicrobial agent. The wafers are capable of sustained in vitro release of bioactive silver for at least 4 weeks. The wafers exhibit silver release that follows erosion kinetics, confirming a bulk erosion/release mechanism. In clinical evaluation, sustained release of silver at bactericidal levels for at least 21 days is observed. Staining of hard and soft tissues due to the released silver is minimal and reversible.

The 3'-5' exonuclease site of DNA polymerase III from gram-positive bacteria: definition of a novel motif structure.

The primary structure of the 3'-5' exonuclease (Exo) site of the Gram+ bacterial DNA polymerase III (Pol III) was examined by site-directed mutagenesis of Bacillus subtilis Pol III (BsPol III). It was found to differ significantly from the conventional three-motif substructure established for the Exo site of DNA polymerase I of Escherichia coli (EcPol I) and the majority of other DNA polymerase-exonucleases. Motifs I and II were conventionally organized and anchored functionally by the predicted carboxylate residues. However, the conventional downstream motif, motif III, was replaced by motif III epsilon, a novel 55-amino-acid (aa) segment incorporating three essential aa (His565, Asp533 and Asp570) which are strictly conserved in three Gram+ Pol III and in the Ec Exo epsilon (epsilon). Despite its unique substructure, the Gram+ Pol III-specific Exo site was conventionally independent of Pol, the site of 2'-deoxyribonucleoside 5-triphosphate (dNTP) binding and polymerization. The entire Exo site, including motif III epsilon, could be deleted without profoundly affecting the enzyme's capacity to polymerize dNTPs. Conversely, Pol and all other sequences downstream of the Exo site could be deleted with little apparent effect on Exo activity. Whether the three essential aa within the unique motif III epsilon substructure participate in the conventional two-metal-ion mechanism elucidated for the model Exo site of EcPol I, remains to be established.

DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC.

Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorously investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.

A single mutation in bacteriophage T4 DNA polymerase (A737V, tsL141) decreases its processivity as a polymerase and increases its processivity as a 3'-->5' exonuclease.

The bacteriophage T4 DNA polymerase mutant A737V (tsL141 and tsCB120) was originally characterized as temperature-sensitive for DNA replication and an antimutator for transition mutations. Its antimutator phenotype is suppressed by the L771F mutation (Reha-Krantz, L. J., Stocki, S., Nonay, R., and Maughan, C. (1989) J. Cell. Biochem. 13D, 140). We find that the A737V polymerase arrests much more frequently than the wild type when polymerizing on primed single-stranded DNA templates. Although the 3'-->5' exonuclease of the mutant is indistinguishable from the wild type on single-stranded DNA, it is more active than the wild type on duplex DNA. In a single encounter with the primer, the wild type polymerase can incorporate more than 50 nucleotides. The processivity of the A737V polymerase is less than the wild type as a polymerase, but is greater than the wild type as an exonuclease. The L771F polymerase resembles the wild type in each of these properties, while the double mutant (A737V, L771F) is intermediate between the two single mutants. Kinetic studies of wild type T4 DNA polymerase (Capson, T. L., Peliska, J. A., Kaboord, B. F., Frey, M. W., Lively, C., Dahlberg, M., and Benkovic, S. J. (1992) Biochemistry 31, 10984-10994) suggest that DNA binds first to the polumerase active site, before adopting a configuration in which it can be hydrolyzed by the exonuclease. Within this framework, our studies suggest that DNA moves more readily from the polymerase- to the exonuclease-competent configuration on the A737V mutant polymerase, and that this movement is decreased by the compensating L771F mutation.

Interaction of DNA polymerase and DNA helicase within the bacteriophage T4 DNA replication complex. Leading strand synthesis by the T4 DNA polymerase mutant A737V (tsL141) requires the T4 gene 59 helicase assembly protein.

The bacteriophage T4 tsL141 (A737V) mutant in T4 DNA polymerase is temperature-sensitive for DNA replication and an antimutator for some types of mutations. In the accompanying paper (Spacciapoli, P., and Nossal, N. G. (1993) J. Biol. Chem. 269, 438-446), we show that the purified A737V T4 DNA polymerase is less processive than the wild type enzyme as a polymerase, but is more processive as an exonuclease. The bacteriophage T4 multienzyme replication complex reconstituted with the A737V mutant polymerase is defective in both lagging and leading strand synthesis. On lagging strand templates, the A737V polymerase is stimulated by the gene 44/62 and 45 polymerase accessory proteins and the gene 32 DNA binding protein, but is still arrested at pause sites much more frequently than the wild type. In contrast to wild type T4 DNA polymerase, the A737V polymerase does not catalyze leading strand synthesis on a forked duplex template with the polymerase accessory proteins, 32 protein, and the gene 41 protein helicase. The A737V polymerase requires the T4 gene 59 helicase assembly protein, as well as the other proteins, to carry out this reaction. Each of these defects is suppressed by the intragenic L771F mutation that suppresses the antimutator phenotype of the A737V, polymerase in vivo (Reha-Krantz, L. J., Stocki, S., Nonay, R., and Maughan, C. (1989) J. Cell. Biochem. 13D, 140).

Structural and catalytic properties of copper in lysyl oxidase.

The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.

Purines in tRNAs required for recognition by ATP/CTP:tRNA nucleotidyltransferase from rabbit liver.

Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase from rabbit liver was studied using 12 tRNAs, previously treated with the chemical modifier diethylpyrocarbonate (DEP). Such chemically modified tRNAs were labeled with 32P by nucleotidyltransferase, using alpha-[32P]ATP as a cosubstrate. A carbethoxylated purine at position 57 in the psi-loop interfered with recognition of the tRNA in all instances. DEP-modified purines at other positions (58 in the psi-loop, 52 or 53 in the psi-stem, and 71-73 in the acceptor stem), also interfered with the interaction, but in only a few tRNAs. The mammalian enzyme was more similar to the homologous enzyme from yeast than that from bacteria, in its requirements for chemically unmodified purines. The extent of exclusion of modified bases from 32P-labeled material diminished as the concentration of enzyme increased, demonstrating that interference was not due to the inability of the chemically altered tRNA to refold into a recognizable conformation. The degree of purification of the enzyme did not affect the identity of bases that inhibited the reaction when modified.

Recognition of tRNA by the enzyme ATP/CTP:tRNA nucleotidyltransferase. Interference by nucleotides modified with diethyl pyrocarbonate or hydrazine.

Treatment of tRNA with diethyl pyrocarbonate or hydrazine prior to incubation with the enzyme ATP/CTP:tRNA nucleotidyltransferase and [alpha-32P]ATP results in exclusion of modified bases from labeled molecules. Purines modified with diethyl pyrocarbonate, which interfere with enzyme recognition, cluster at the corner of the tRNA molecule, where the D- and psi-loops are juxtaposed in all 15 tRNAs used in this study. When the enzyme is isolated from Escherichia coli, few other sites of interference are evident near the 3'-end; when the homologous enzyme from yeast is used, more exclusions are apparent near the 3'-end. Modification of uridines with hydrazine has no effect on interaction with the enzyme, except for one uridine near the 3'-end of tRNA(Gly). Interference of enzyme activity by modified bases can be overcome by longer incubation times or increased concentrations of enzyme.