Bioinformatic investigation of the cost management strategies of five oral microbes.

Some amino acids are more energetically costly to synthesize de novo, therefore many microbes have evolved to regulate the metabolic expenditure of the cell and reduce the energy burden of extracellular unrecyclable proteins. Several oral bacterial species take up amino acids and peptides obtained from proteolysis of host proteins and hence do not rely only on de novo synthesis. The aim of this study was to investigate if five oral bacterial species implement cost management strategies to reduce the energy burden of extracellular unrecyclable proteins. Since the relative de novo amino acid synthesis costs are proportional to the masses of the amino acids, the energy costs of producing proteins were assessed by calculating the mean amino acid mass for each protein. For Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia and Streptococcus sanguinis, the outer membrane/extracellular proteins are made up of a much larger percentage of lower average mass amino acids whereas cytoplasmic proteins are made up of a larger proportion of higher average mass amino acid residues. These results are consistent with the five oral bacterial species employing energy-saving mechanisms in the production of extracellular unrecyclable proteins. Interestingly, the P. gingivalis and S. sanguinis genomes exhibited significantly lower predicted mean amino acid masses compared with those of the genomes of the other three species, suggesting that this may provide them with an energy advantage with respect to protein biosynthetic cost.

Optical fiber imaging for high speed plasma motion diagnostics: applied to low voltage circuit breakers.

An integrated portable measurement system is described for the study of high speed and high temperature unsteady plasma flows such as those found in the vicinity of high current switching arcs. An array of optical fibers allows the formation of low spatial resolution images, with a maximum capture rate of 1 x 10(6) images per second (1 MHz), with 8 bit intensity resolution. Novel software techniques are reported to allow imaging of the arc; and to measure arc trajectories. Results are presented on high current (2 kA) discharge events in a model test fixture and on the application to a commercial low voltage circuit breaker.

Enamel subsurface lesion remineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and fluoride.

Casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) and amorphous calcium fluoride phosphate (CPP-ACFP) solutions have been shown to remineralise enamel subsurface lesions. The aim of this study was to determine the effect of ion composition of CPP-ACP and CPP-ACFP solutions on enamel subsurface lesion remineralisation in vitro. CPP-bound and free calcium, phosphate and fluoride ion concentrations in the solutions were determined after ultrafiltration. The ion activities of the free ion species present were calculated using an iterative computational program. The mineral deposited in the subsurface lesions was analysed using transverse microradiography and electron microprobe. CPP was found to stabilise high concentrations of calcium, phosphate and fluoride ions at all pH values (7.0-4.5). Remineralisation of the subsurface lesions was observed at all pH values tested with a maximum at pH 5.5. The CPP-ACFP solutions produced greater remineralisation than the CPP-ACP solutions at pH 5.5 and below. The mineral formed in the subsurface lesions was consistent with hydroxyapatite and fluorapatite for remineralisation with CPP-ACP and CPP-ACFP, respectively. The activity gradient of the neutral ion pair CaHPO(4)(0) into the lesion was significantly correlated with remineralisation and together with HF(0) were identified as important species for diffusion.

Effect of addition of citric acid and casein phosphopeptide-amorphous calcium phosphate to a sugar-free chewing gum on enamel remineralization in situ.

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) has been shown to remineralize enamel subsurface lesions in situ. The aim of this study was to investigate the effects of CPP-ACP in a fruit-flavoured sugar-free chewing gum containing citric acid on enamel remineralization, and acid resistance of the remineralized enamel, using an in situ remineralization model. The study utilized a double-blind, randomized, crossover design with three treatments: (i) sugar-free gum (2 pellets) containing 20 mg citric acid and 18.8 mg CPP-ACP, (ii) sugar-free gum containing 20 mg citric acid alone, (iii) sugar-free gum not containing CPP-ACP or citric acid. Ten subjects were instructed to wear removable palatal appliances, with 4 half-slab insets of human enamel containing demineralized subsurface lesions and to chew gum (2 pellets) for 20 min 4 times per day for 14 days. At the completion of each treatment the enamel half-slabs were removed and half of the remineralized lesion treated with demineralization buffer for 16 h in vitro. The enamel slabs (remineralized, acid-challenged and control) were then embedded, sectioned and subjected to microradiography to determine the level of remineralization. Chewing with gum containing citric acid and CPP-ACP resulted in significantly higher remineralization (13.0 +/- 2.2%) than chewing with either gum containing no CPP-ACP or citric acid (9.4 +/- 1.2%) or gum containing citric acid alone (2.6 +/- 1.3%). The acid challenge of the remineralized lesions showed that the level of mineral after acid challenge was significantly greater for the lesions exposed to the gum containing CPP-ACP.

Casein phosphopeptides in oral health--chemistry and clinical applications.

The casein phosphopeptides (CPP) are derived from the milk protein casein by tryptic digestion. The CPP, containing the sequence -Pse-Pse-Pse-Glu-Glu- where Pse is a phosphoseryl residue, stabilize calcium and phosphate ions in aqueous solution and make these essential nutrients bioavailable. Under alkaline conditions the calcium phosphate is present as an alkaline amorphous phase complexed by the CPP, referred to as casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). The CPP-ACP complexes readily incorporate fluoride ions forming casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP). A mechanism is discussed which provides a rationale for the ability of the CPP-ACP to remineralize carious lesions in dental enamel. Clinical applications of the CPP-ACP as agents in the treatment of dental caries and other hypomineralized conditions are reviewed. It is concluded that the CPP are a safe and novel carrier for calcium, phosphate and hydroxide (fluoride) ions to promote enamel remineralization with application in oral care products, dental professional products and foodstuffs.

Protein dynamics of bovine dentin phosphophoryn.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and o-phosphoseryl [Ser(P)] residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentin. The elucidation of the structure-function relationship of phosphophoryn has been a challenge because of its high-molecular weight, high negative charge, repetitive sequence, and lability. We have used the dynamic behavior of the (1)H NMR signal at 600 MHz to provide insight into the molecular dynamics of phosphophoryn. Our results indicate that phosphophoryn is a molecule of uniformly high mobility, thus belonging to a recently identified class of intrinsically disordered proteins that are characterized by sequences of low complexity and rich in polar and charged residues. The significance of our results is that phosphophoryn, because of its uniform nature has the potential to be replaced by biomimetic synthetic peptide analogs that together with amorphous calcium phosphate may lead to the development of novel, nontoxic, apatite-based dental restorative materials.

NMR studies of a novel calcium, phosphate and fluoride delivery vehicle-alpha(S1)-casein(59-79) by stabilized amorphous calcium fluoride phosphate nanocomplexes.

The repair of early tooth enamel lesions has been recently demonstrated by tryptic phosphopeptides derived from milk caseins that associate with amorphous calcium phosphate (ACP) forming stable complexes. These casein phosphopeptides (CPP), containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, form calcium phosphate delivery vehicles that retard enamel demineralization and promote remineralization. Recently, we have shown that these peptides also stabilize calcium fluoride phosphate as soluble complexes. These complexes designated CPP-ACFP, have the potential to provide superior clinical efficacy in preventing dental caries and treating and repairing early stages of disease. In an approach to determine the ultrastructure of the casein phosphopeptide-amorphous calcium fluoride phosphate complexes, we have studied the structure of the predominant peptide alpha(S1)-CN(59-79) bound to ACFP using nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. The alpha(S1)-CN(59-79) peptide stabilized calcium fluoride phosphate as amorphous nanocomplexes with a hydrodynamic radius of 2.12+/-0.26 nm. The nanocomplexes exhibited stoichiometry of one peptide to 15 calcium, nine phosphate and three fluoride ions. Sequence-specific resonance assignments were determined for the peptide alpha(S1)-CN(59-79) complexed to the ACFP. The secondary structure of the peptide alpha(S1)-CN(59-79) was characterized by sequential (i, i+1), medium-range (i, i+2) nOes and H alpha chemical shifts. The spectral data were compared with that of the peptide alpha(S1)-CN(59-79) bound to calcium ions, revealing that the structurally significant secondary NH and alpha-chemical shifts were similar.

Hemoglobin hydrolysis and heme acquisition by Porphyromonas gingivalis.

Porphyromonas gingivalis has been implicated in the progression of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. This bacterium is a gram-negative, black-pigmented, asaccharolytic anaerobe that relies on the fermentation of amino acids for the production of metabolic energy. The Arg- and Lys-specific extracellular cysteine proteinases of P. gingivalis, RgpA, RgpB and Kgp have been implicated as major virulence factors. In this study we investigated the hydrolysis of human hemoglobin by whole cells of P. gingivalis W50 and the mutants W501 (RgpA-), W50AB (RgpA-RgpB-) and W50ABK (RgpA-RgpB-Kgp-) under strictly anaerobic conditions in a physiological buffer (pH 7.5) using mass spectrometric analysis. Incubation of P. gingivalis W50 with hemoglobin over a period of 30 min resulted in the detection of 20 hemoglobin peptides, all with C-terminal Arg or Lys residues. The majority of the hemoglobin alpha- and beta-chain sequences were recovered as peptides except for two similar regions of the C-terminal half of each chain, alpha(92-127) and beta(83-120). The residues of the unrecovered sequences form part of the interface between the alpha- and beta-chains and an exposed surface area of the hemoglobin tetramer that may be involved in binding to P. gingivalis. P. gingivalis W501 (RgpA-) produced similar peptides to those seen in the wild-type. All identified peptides from the hydrolysis of hemoglobin by the P. gingivalis W50AB (RgpA-RgpB-) mutant were the result of cleavage at Lys. The triple mutant W50ABK was unable to hydrolyze hemoglobin under the assay conditions used, suggesting that on whole cells the major cell surface activity responsible for hydrolysis of hemoglobin is from the RgpA/B and Kgp proteinases. However, the triple proteinase mutant W50ABK grew as well as the wild-type in a medium containing hemoglobin as the only iron source, indicating that the RgpA/B and Kgp proteinases are not essential for iron assimilation from hemoglobin by P. gingivalis.

Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics.

Influenza haemagglutinin (HA) is responsible for fusing viral and endosomal membranes during virus entry. In this process, conformational changes in the HA relocate the HA(2) N-terminal 'fusion peptide' to interact with the target membrane. The highly conserved HA fusion peptide shares composition and sequence features with functionally analogous regions of other viral fusion proteins, including the presence and distribution of glycines and large side-chain hydrophobic residues. HAs with mutations in the fusion peptide were expressed using vaccinia virus recombinants to examine the requirement for fusion of specific hydrophobic residues and the significance of glycine spacing. Mutant HAs were also incorporated into infectious influenza viruses for analysis of their effects on infectivity and replication. In most cases alanine, but not glycine substitutions for the large hydrophobic residues, yielded fusion-competent HAs and infectious viruses, suggesting that the conserved spacing of glycines may be structurally significant. When viruses containing alanine substitutions for large hydrophobic residues were passaged, pseudoreversion to valine was observed, indicating a preference for large hydrophobic residues at specific positions. Viruses were also obtained with serine, leucine or phenylalanine as the N-terminal residue, but these replicated to significantly lower levels than wild-type virus with glycine at this position.

Kappacin, a novel antibacterial peptide from bovine milk.

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk kappa-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated kappa-casein (residues 106 to 169) [kappa-casein(106--169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)(149)kappa-casein-A(138--158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)(149)kappa-casein-A(138--158) and its nonphosphorylated counterpart kappa-casein-A(138--158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)(149) kappa-casein-A(138--158) displayed growth-inhibitory activity against S. mutans (MIC, 59 microg/ml [26 microM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.

Cation-dependent structural features of beta-casein-(1-25).

Complete sequence-specific, proton-resonance assignments have been determined for the calcium phosphate-stabilizing tryptic peptide beta-casein-(1-25) containing the phosphorylated sequence motif Ser(P)(17)-Ser(P)-Ser(P)-Glu-Glu(21). Spectra of the peptide have been recorded, in separate experiments, in the presence of excess ammonium ions, sodium ions and calcium ions, and of the dephosphorylated peptide in the presence of excess sodium ions. We observed significant changes to chemical shifts for backbone and side-chain resonances that were dependent upon the nature of the cation present. Medium-range nuclear Overhauser effect (nOe) enhancements, characteristic of small structured regions in the peptide, were observed and also found to be cation dependent. The secondary structure of the peptide was characterized by sequential and medium-range (i, i+2/3/4, which denotes an interaction between residue i and residue i+2, i+3 or i+4 in the peptide) nOe connectivities, and Halpha chemical shifts. Four structured regions were identified in the calcium-bound peptide: residues Arg(1) to Glu(4) were involved in a loop-type structure, and residues Val(8) to Glu(11), Ser(P)(17) to Glu(20) and Glu(21) to Thr(24) were implicated in beta-turn conformations. Comparison of the patterns of medium-range nOe connectivities in beta-casein-(1-25) with those in alpha(S1)-casein-(59-79) suggest that the two peptides have distinctly different conformations in the presence of calcium ions, despite having a high degree of sequential and functional similarity.

Mechanisms of cell entry by influenza virus.

A wide range of viruses, including many human and animal pathogens representing various taxonomic groups, contain genomes that are enclosed in lipid envelopes. These envelopes are generally acquired in the final stages of assembly, as viruses bud from regions of the membrane of the infected cell at which virally encoded membrane proteins have accumulated. The viruses procure their membranes during this process and mature particles 'pinch off' from the cellular membranes. Under most circumstances, initiation of another round of infection is dependent on two critical functions supplied by the envelope proteins. The virus must bind to cell-surface receptors of a new host cell, and fusion of the viral and cellular membranes must occur to transfer the viral genome into the cell. Enveloped viruses have evolved a variety of mechanisms to execute these two basic functions. Owing to their relative simplicity, studies of binding and fusion using enveloped viruses and their components have contributed significantly to the overall understanding of receptor-ligand interactions and membrane fusion processes - fundamental activities involved in a plethora of biological functions.

Characterization of a novel outer membrane hemin-binding protein of Porphyromonas gingivalis.

Porphyromonas gingivalis is a gram-negative, anaerobic coccobacillus that has been implicated as a major etiological agent in the development of chronic periodontitis. In this paper, we report the characterization of a protein, IhtB (iron heme transport; formerly designated Pga30), that is an outer membrane hemin-binding protein potentially involved in iron assimilation by P. gingivalis. IhtB was localized to the cell surface of P. gingivalis by Western blot analysis of a Sarkosyl-insoluble outer membrane preparation and by immunocytochemical staining of whole cells using IhtB peptide-specific antisera. The protein, released from the cell surface, was shown to bind to hemin using hemin-agarose. The growth of heme-limited, but not heme-replete, P. gingivalis cells was inhibited by preincubation with IhtB peptide-specific antisera. The ihtB gene was located between an open reading frame encoding a putative TonB-linked outer membrane receptor and three open reading frames that have sequence similarity to ATP binding cassette transport system operons in other bacteria. Analysis of the deduced amino acid sequence of IhtB showed significant similarity to the Salmonella typhimurium protein CbiK, a cobalt chelatase that is structurally related to the ATP-independent family of ferrochelatases. Molecular modeling indicated that the IhtB amino acid sequence could be threaded onto the CbiK fold with the IhtB structural model containing the active-site residues critical for chelatase activity. These results suggest that IhtB is a peripheral outer membrane chelatase that may remove iron from heme prior to uptake by P. gingivalis.

N-terminal sequence analysis of bovine dentin phosphophoryn after conversion of phosphoseryl to S-propylcysteinyl residues.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser[P]) residues, is synthesized by odontoblasts and is believed to be involved in matrix-mediated biomineralization of dentin. We have purified BDP, using selective precipitation and ion exchange chromatography, from an EDTA soluble dentin extract and converted the Ser(P) residues to S-propylcysteinyl residues that are stable to Edman degradation, facilitating the determination of the amino acid sequence of the N-terminal 38 residues. After the initial Asp-Ser(P)-Pro-Asn-Ser(P)-Ser(P)-Asp-Glu-Ser(P)-Asn-Gly-, the sequence contained the repeated motifs Asp-Ser(P) and Asp-Ser(P)-Ser(P). Purified BDP migrated as a single band on gradient SDS-PAGE with an apparent molecular weight of 156 kDa. This value was consistent with the molecular weight of the dephosphorylated protein of 105 kDa determined by means of MALDI mass spectrometry.

A 1H-NMR study of the casein phosphopeptide alpha s1-casein(59-79).

Complete sequence-specific resonance assignments have been determined for a calcium phosphate sequestering, phosphoseryl-containing, tryptic peptide alpha s1-casein(59-79) containing the phosphorylated motif -SSSEE-. Spectra have been recorded in the presence of excess Ca2+ and at three different values of sample pH to characterize the changes in peptide conformation as calcium binds to the phosphorylated residues. The secondary structure of the peptide was characterized by sequential (i,i + 1), medium-range (i,i + 2/3/4), and long-range (i,i + 5) NOE connectivities, C alpha H chemical shifts, NH to C alpha H coupling constants and the observation of slowly exchanging amide protons. Two structured regions have been identified: residues P73 to V76 implicated in beta-turn conformations, and residues E61 to sigma 67 involved in a loop-type structure.

A 1H-NMR determination of the solution structure of the A-chain of insulin: comparison with the crystal structure and an examination of the role of solvent.

The 1H-NMR chemical shift assignments for the oxidized A-chain of bovine insulin have been determined in aqueous and 30% trifluoroethanol/water solutions. Analysis of the observed medium-range nuclear Overhauser effects indicates that in aqueous solution significant populations of the peptide exist, with a 3(10)-helical conformation over residues 12-17. This region corresponds to helix A (13-20) in the crystal structure of the 2 Zn insulin hexamer. In 30% TFE solution, the NOE data are supportive of a random coil conformation throughout the peptide.

Selection by site-directed antibodies of small regions of peptides which are ordered in water.

High resolution nuclear magnetic resonance has been used to study a short peptide which corresponds to the antigenic region of a larger peptide immunogen. This work shows that there is a strong conformational preference for a Type II beta-turn in solution. This observation has implications for the nature of the immunogenicity and antigenicity of peptide antigens as well as for the more general question of protein-folding mechanisms.

The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn.

Many short synthetic peptides have now been shown to induce antibodies reactive with their cognate sequences in the intact folded protein. Aside from the usefulness of such antibodies as site-specific reagents, the frequency with which this recognition occurs has raised several theoretical issues, the central one being that of how an antibody to a short synthetic peptide, which represents one of the most disordered states of a site in a protein, can react with the more ordered version of the same sequence in the folded protein. This apparent paradox can be resolved if the target site on the protein approaches disorder or if the peptide in solution or on a carrier adopts, with significant frequency, a conformation compatible with that of the cognate site in the protein. Various studies already suggest that antigenic sites in proteins correspond to regions of high atomic mobility. We now show, using high-field nuclear magnetic resonance (NMR) spectroscopy, that a nonapeptide selected by several monoclonal antibodies as the immunodominant site of a 36-amino-acid immunogen (residues 75-110 of influenza virus haemagglutinin) adopts a highly populated type-II reverse-turn conformation in water. This suggests that in this case the antibodies have selected a sequence possessing a conformational preference. Apart from helping us to understand immunological recognition, anti-peptide antibodies may provide reagents of sufficient precision for an immunological approach to the problem of protein folding.

Assignment of acyl chain resonances from membranes of mammalian cells by two-dimensional NMR methods.

Two-dimensional nuclear magnetic resonance (NMR) methods have been successfully used to assign resonances in the 1H NMR spectrum of intact viable rat mammary adenocarcinoma cells. Two-dimensional scalar-correlated spectroscopy identifies connectivities for resonances of the lipid acyl chains in the plasma membrane of these cells. We expect that two-dimensional scalar-correlated methods may be of general use for providing unequivocal assignments in the complex and often poorly resolved 1H NMR spectra of cells.