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Objectives: To compare the remineralisation efficacy and ion bioavailability of two novel SnF 2 -containing dentifrices in a blinded, cross-over, randomised in situ clinical study., Methods: Six participants wore removal palatal appliances holding human enamel and dentine blocks with subsurface lesions. Appliances were worn for two treatment periods of 14 consecutive days each, with a one-week washout period in-between. Participants were randomly allocated to rinse with a 1:5 diluted coded slurry of one of two dentifrices containing either 5 % casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) +1100 ppm F as SnF 2 [MIPOP], or 1100 ppm F as SnF 2 [CT], for 1 min, four times a day. Saliva was collected post-treatment and analysed for tin, calcium, inorganic phosphate and fluoride ions using atomic absorption spectrophotometry and ion chromatography. Enamel and dentine lesions were analysed for percent remineralisation (%R) using transverse microradiography and percent surface microhardness recovery (%SMHR)., Results: MIPOP released significantly higher F (3.00 ± 0.27 mM), Ca (15.23 ± 3.23 mM) and Sn (1.18 ± 0.13 mM) into saliva whereas CT released 2.89 ± 0.32 mM F and only 0.84 ± 0.11 mM Ca and 0.28 ± 0.10 mM Sn. MIPOP produced significantly higher %R than CT: 25.6 ± 1.5 % compared to 15.2 ± 0.7 % in enamel, and 33.6 ± 3.1 % compared to 20.6 ± 1.1 % in dentine. Additionally, MIPOP produced significantly higher %SMHR (18.2 ± 7.9 %) compared to CT (4.1 ± 0.6 %)., Conclusions: Both dentifrices promoted remineralisation, but the MIPOP dentifrice with added CPP-ACP and the ion-stabilising effects of CPP released higher amounts of bioavailable tin and produced significantly higher remineralisation and surface microhardness recovery., Clinical Significance: Modern dentifrices contain SnF 2 for a range of oral health benefits. Challenges associated with stability of these formulations can affect ion bioavailability, reducing efficacy. Two dentifrices with SnF 2 promoted remineralisation in situ, however the dentifrice with the added saliva biomimetic CPP-ACP was superior and therefore may produce greater health benefits., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: The aim of this study was to investigate the capacity of an approved food additive with anticariogenic properties, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), when added to a processed, sugar-containing yogurt with added lactic-acid bacteria (probiotics), to prevent demineralization of enamel subsurface lesions in vitro., Methods: Enamel subsurface demineralised lesions were created in thirty extracted human third molars. These were then exposed to artificial saliva (AS) with: 1) Danone yogurt alone; 2) Danone yogurt with 0.2 % w/w CPP-ACP; or 3) Danone yogurt with 0.5 % w/w CPP-ACP at 37 °C for two weeks. The yogurt/AS was replaced with fresh preparations each day. At the completion of each treatment the enamel slabs were embedded, sectioned and analyzed using transverse microradiography to measure changes in enamel lesion depths and subsurface mineral content. Yogurt samples were analysed for soluble calcium (Ca) and inorganic phosphate (Pi) levels and pH., Results: Yogurt alone demineralized enamel subsurface lesions to produce significantly larger lesions. However, the addition of 0.2 % CPP-ACP to the yogurt resulted in significant reduction in demineralization compared with yogurt alone (p < 0.0001). The addition of 0.5 % CPP-ACP to the yogurt produced a net remineralization effect with a significant increase in lesion mineral content (p < 0.0001). The addition of CPP-ACP resulted in a significant (p < 0.0001) dose-related increase in Ca, Pi and pH., Conclusions: The addition of CPP-ACP to a commercial yogurt exhibited a dose related protective effect with 0.5 % CPP-ACP producing remineralization of existing enamel subsurface lesions under the in vitro experimental conditions. CLINICAL SIGNIfiCANCE: The results of this study suggest that some processed yogurts with added sugar could result in enamel demineralization when frequently consumed by individuals with poor oral hygiene. The addition of CPP-ACP to these yogurts may help prevent demineralization and promote enamel subsurface lesion remineralization, and therefore, make them safer for teeth., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: To determine if chewing gum containing casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) promoted an increase in the abundance of Streptococcus sanguinis and other species associated with dental health in supragingival plaque in a clinical study., Materials and Methods: Nineteen participants were recruited for a three-leg cross-over, randomised, controlled clinical trial. Participants chewed a sugar-free gum with or without CPP-ACP six times daily for 20 min over two weeks. The study also involved no gum chewing (no gum) for the same two week period. Participants were randomly assigned to one of the test gums or no gum for each intervention period. Participants abstained from oral hygiene and had washout periods of two weeks between intervention periods. After each intervention period, supragingival plaque was collected and analysed for bacterial composition by sequencing the V4 variable region of the 16S rRNA gene. Data were analysed using a linear mixed model., Results: The CPP-ACP gum intervention produced a significant (p < 0.01) increase in the proportions of S. sanguinis (112%), as well as the commensal species Rothia dentocariosa (127%), Corynebacterium durum (80%) and Streptococcus mitis (55%) when compared with the no gum intervention. All the species that were promoted by the CPP-ACP gum are known to possess one or both of the alkali-producing enzymes arginine deiminase and nitrate reductase., Conclusion: This clinical study demonstrated that chewing a sugar-free gum containing CPP-ACP promoted prebiosis by significantly increasing the proportion of S. sanguinis and other health-associated bacterial species in supragingival plaque., Clinical Significance: Regular chewing of CPP-ACP sugar-free gum increases the proportions of health-associated commensal species in supragingival plaque to promote prebiosis and oral homeostasis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Soy beverages are promoted as healthy alternatives to bovine milk even though they can contain added sugar., Objectives: To compare enamel mineral content after consumption of bovine milk or a soy beverage in a double-blind, randomized, cross-over in situ clinical study., Materials and Methods: Human enamel slabs with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers who consumed 200 ml of either bovine milk or a soy beverage over a 60 s period once a day for 15 days. Enamel lesion depth and mineral content were measured using transverse microradiography. Saliva samples were collected immediately after consuming the beverages and calcium, inorganic phosphate and fluoride levels analysed. Data were statistically analysed using a linear mixed model., Results: Depth of the enamel subsurface lesions increased by 7.1 ± 2.0 μm and mineral content decreased by 47 ± 22 vol% min.μm after consumption of the soy beverage indicating demineralization. However, after consumption of bovine milk the depth of the lesions decreased by 7.6 ± 3.5 μm and mineral content increased by 202 ± 43 vol% min.μm indicating remineralization. The changes were significantly different (p < 0.001) between the two beverages. Fluoride levels were similar in the saliva samples for both beverages, however the calcium and inorganic phosphate levels for the bovine milk group were significantly higher (p < 0.02) than those for the soy beverage group., Conclusions: In this randomized, double-blind in situ clinical trial consumption of a soy beverage demineralized enamel whereas bovine milk produced remineralization., Clinical Significance: Although soy beverages are promoted as healthy alternatives to bovine milk the added sugar and low calcium bioavailability of the soy drink makes frequent consumption a caries risk. (Trial registration no. ISRCTN19137849)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objectives: To compare remineralization of enamel subsurface lesions by fluoride dentifrices with added calcium in a double-blind, randomized, cross-over, in situ study., Methods: Human enamel with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers. A slurry (1 g toothpaste/4 ml H 2 O) was rinsed for 60 s, 4 times per day for 14 days. Seven toothpastes were tested: (i) 1450 ppm F (NaF), (ii) 5000 ppm F (NaF), (iii) 1450 ppm F (MFP) with calcium sodium phosphosilicate (CSP), (iv) 1450 ppm F (MFP) with CaCO 3 /Arg, (v) 1150 ppm F (SnF 2 ) with amorphous calcium phosphate (ACP), (vi) 1100 ppm F (NaF) with casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and (vii) 5000 ppm F (NaF) with functionalized tri-calcium phosphate (TCP). Total (acid soluble) and bioavailable (water soluble) calcium, inorganic phosphate and fluoride levels of the dentifrices were measured using ion chromatography (F/MFP) and spectrophotometry (Ca and inorganic phosphate). Enamel lesion mineral content was measured using transverse microradiography. Data were statistically analysed using a linear mixed model., Results: All calcium and fluoride containing toothpastes released > 90% of bioavailable fluoride and were superior to the respective fluoride alone toothpastes in remineralization of enamel subsurface lesions. The level of remineralization followed the order: CPP-ACP/1l00 ppm F > ACP/1150 ppm F = TCP/5000 ppm F > 5000 ppm F = CaCO 3 /Arg/1450 ppm F = CSP/1450 ppm F > 1450 ppm F. Bioavailable calcium levels significantly correlated with enhanced remineralization of enamel subsurface lesions., Conclusions: Bioavailable calcium in fluoride dentifrices enhanced remineralization of enamel subsurface lesions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Viruses impose an immense burden on human health. With the goal of treating and preventing viral infections, researchers have carried out genetic screens to improve our understanding of viral dependencies and identify potential anti-viral strategies. The emergence of CRISPR genetic screening tools has facilitated this effort by enabling host-virus screens to be undertaken in a more versatile and fidelitous manner than previously possible. Here we review the growing number of CRISPR screens which continue to increase our understanding of host-virus interactions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and CPP-ACP with fluoride (CPP-ACFP) have been shown to provide bioavailable ions to promote mineralization. Hence, the aim of this study was to evaluate the materials' biocompatibility and osteogenic/calcification potential for endodontic applications., Methods: Human and mouse osteoblast-like and fibroblast-like cell lines were incubated with 0.05%-3.0% w/v CPP-ACP and CPP-ACFP, and toxicity, proliferation, alkaline phosphatase, interleukin (IL)-1α, and IL-6 production, collagen type I, osteocalcin, and osteopontin production, and mineralization/calcification were determined., Results: CPP-ACP and CPP-ACFP were non-toxic and had no significant effect on proliferation or production of the inflammatory cytokine IL-1α. Alkaline phosphatase activity of the osteoblast-like cells was significantly increased (P < .05) by CPP-ACP and CPP-ACFP, as was the production of the osteotropic cytokine IL-6, the formation of calcium mineral deposits, and the secretion of mineralization-related proteins (collagen type I and osteocalcin)., Conclusions: CPP-ACP and CPP-ACFP are biocompatible and have the potential to induce osteoblastic differentiation and mineralization. Potential applications include apexification, perforation repair, vital pulp therapy, and regenerative endodontic procedures., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Sugar-free chewing gum containing polyols has been demonstrated to reduce caries experience in randomised controlled clinical trials. A range of polyols (mannitol, sorbitol, xylitol and maltitol) can be found in sugar-free gums and it has been claimed that they can facilitate calcium uptake into enamel subsurface lesions promoting remineralisation., Objectives: The aim of this study was to compare the effect of polyols on remineralisation of enamel subsurface lesions in vitro by artificial saliva (AS) and by AS containing the salivary biomimetic casein phosphopeptide amorphous calcium phosphate (CPP-ACP)., Methods: The polyols (12.6% w/v) and CPP-ACP (0.376% w/v) were used at physiologically relevant concentrations approximating those released into saliva during chewing a CPP-ACP/polyol chewing gum. Enamel subsurface lesions were exposed to one of the polyols (xylitol, sorbitol, maltitol, mannitol) in AS or AS containing CPP-ACP for 7days at 37°C with a change of solution each day. Remineralisation of the enamel subsurface lesions was measured by transverse microradiography., Results: A statistical test for equivalence showed there was no difference in remineralisation between the AS solutions with or without any of the polyols. The AS+CPP-ACP solution substantially promoted remineralisation over AS alone independently of any polyol added., Conclusion: This controlled in vitro study showed that polyols at physiologically relevant concentrations did not promote remineralisation of enamel subsurface lesions by facilitating calcium uptake into the lesion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: Internal bleaching of discolored teeth uses sodium perborate reacting with water to form the active agent, hydrogen peroxide (H 2 O 2 ). Sodium perborate is replaced at varying time intervals depending on clinician preference and until esthetically acceptable results are achieved, but this is done without scientific basis. This study measured the depletion rate of hydrogen peroxide from sodium perborate as a bleaching agent., Methods: Two sodium perborate bleaching products (Odontobleach [Australian Dental Manufacturing, Kenmore Hills, Queensland, Australia] and Endosure Perborate Micro [Dentalife, Ringwood, Victoria, Australia]) and distilled deionized water mixtures at ratios of 25 μg/mL, 50 μg/mL, and 100 μg/mL were placed into sealed microtubes and incubated at 37°C. H 2 O 2 concentrations were measured at 23 time points over 4 weeks. Quantification of H 2 O 2 concentrations was obtained using a ferrothiocyanate oxidation reduction reaction followed by spectrophotometry readings., Results: The H 2 O 2 concentration rapidly peaked within 27 hours and reached a plateau by about 3 days (75 hours). Low levels of H 2 O 2 were evident beyond 3 days and for at least 28 days. No significant differences were found between the 2 sodium perborate products. There was also no significant difference in the depletion rate between the different ratios., Conclusions: Based on the chemistry of H 2 O 2 depletion, the minimum replacement interval for the bleaching agent is 3 days. Frequent replacements of the perborate clinically may be unnecessary because of the continued presence of low H 2 O 2 levels for at least 28 days. Although these data cannot be extrapolated to the clinical situation, they set a baseline for further studies to address the many clinical variables influencing internal bleaching., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objectives: This review paper evaluates the use of in vitro biofilm models for the testing of anticariogenic agents., Data: Caries is a biofilm-mediated oral disease and in vitro biofilm models have been widely utilised to assess how anticariogenic or antimicrobial agents affect the de/remineralisation process of caries. The use of enamel or dentine substrata has enabled the assessment of the relationship between bacterial activity and caries lesion initiation and progression and how this relationship could be affected by the agent under study., Sources: Only papers published in the English literature were reviewed., Study Selection: Both 'open' and 'closed' biofilm systems utilising either single or multiple-species as defined or undefined inocula are analysed., Conclusions: There is a wide variety of in vitro biofilm models used in the assessment of anticariogenic agents. A reproducible model that mimics the shear forces present in the oral environment, and uses a defined multiple-species inocula on tooth substrates can provide valuable insight into the effectiveness of these agents., Clinical Relevance: Biofilm models are important tools for the testing of the mechanism of action and efficacy of novel anticariogenic agents. Results from these experiments help facilitate the design of randomised, controlled clinical trials for testing of efficacy of the agents to provide essential scientific evidence for their clinical use., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: To determine the potential acidogenicy of liquid breakfasts., Methods: In vitro acid production by Streptococcus mutans was measured in the beverages at a pH of 5.5, as was the fall in pH over 10min. The buffering capacity was determined, as well as the calcium, inorganic phosphate and fluoride concentrations (total and soluble) of the beverages. Bovine milk (UHT) was used for comparison., Results: The rate of acid production by S. mutans, and pH fall over 10min was greater in liquid breakfasts compared to bovine milk. All beverages except one demonstrated a significantly lower buffering capacity than bovine milk. All beverages contained significantly greater concentrations of soluble calcium than bovine milk, and all except two contained significantly more soluble inorganic phosphate., Conclusions: S. mutans was able to generate significantly more acid in the liquid breakfasts than in bovine milk, indicating these drinks may contribute to a cariogenic diet. In general, the liquid breakfasts required significantly less acid than bovine milk to reduce their pH to the approximate critical pH for enamel demineralisation. However, the liquid breakfasts also tended to contain significantly more soluble calcium and inorganic phosphate than bovine milk., Clinical Significance: The substantial amounts and various types of sugars found within liquid breakfast beverages may result in a significant pH drop in dental plaque following consumption of these products., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a keystone pathogen in the development of the bacterial-associated inflammatory oral disease chronic periodontitis. Although post-translational modifications (PTMs) of proteins are commonly found to modify protein function in eukaryotes and prokaryotes, PTMs such as lysine acetylation have not been examined in P. gingivalis. Lysine acetylation is the addition of an acetyl group to a lysine which removes this amino acid's positive charge and can induce changes in a protein's secondary structure and reactivity. A proteomics based approach combining immune-affinity enrichment with high sensitivity Orbitrap mass spectrometry identified 130 lysine acetylated peptides from 92 P. gingivalis proteins. The majority of these peptides (71) were attributed to 45 proteins with predicted metabolic activity; these proteins could be mapped to several P. gingivalis metabolic pathways where enzymes catalysing sequential reactions within the same pathway were often found acetylated. In particular, the catabolic pathways of complex anaerobic fermentation of amino acids to produce energy had 12 enzymes lysine acetylated. The results suggest that lysine acetylation may be an important mechanism in metabolic regulation in P. gingivalis, which is vital for P. gingivalis survival and adaptation of its metabolism throughout infection. Statement of significance. Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The ability of the pathogen to induce dysbiosis and disease is related to an array of specific virulence factors and metabolic regulation that enables the bacterium to proliferate in an inflamed periodontal pocket. The mechanisms P. gingivalis uses to adapt to a changing and hostile environment are poorly understood and here we show, for the first time, that enzymes of critical metabolic pathways for energy production in this bacterium were acetylated on certain lysine residues. These enzymes were often found catalysing sequential reactions within the same catabolic pathway. The results suggest that lysine acetylation is an important mechanism of metabolic regulation in P. gingivalis vital for its adaptation and proliferation to produce disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objectives: Candida, an opportunistic fungal pathogen, has been implicated in oral and oesophageal cancers. This study aimed to examine oral Candida carriage in 52 oral cancer patients and 104 age-, gender- and denture status-matched oral cancer-free subjects., Material and Methods: We assessed general health, smoking and alcohol drinking habits, use of alcohol-containing mouthwash and periodontal status (community periodontal index of treatment needs). Yeasts were isolated using oral rinse technique and genetically identified via Real-Time PCR-High resolution melting curve analysis of conserved ribosomal DNA. Conditional and binary logistic regressions were used to identify explanatory variables that are risk factors for oral cancer., Results and Conclusion: The frequencies of oral yeasts' presence and high oral colonization were significantly higher in oral cancer than non-oral cancer patients (p=001; p=0.033, respectively). No significant difference in the isolation profile of Candida species was found between the two groups, except C. parapsilosis was more frequent in non-oral cancer group. Differences were noticed in the incidence of C. albicans strains where significantly more C. albicans genotype-A was isolated from cancer patients and significantly more C. albicans genotype-B isolated from non-cancer patients. Multiple regression analyses showed significant association with cancer observed for alcohol drinking (OR=4.253; 95% CI=1.351, 13.386), Candida presence (OR=3.242; 95% CI=1.505, 6.984) and high oral colonization (OR=3.587; 95% CI=1.153, 11.162). These results indicate that there is a significant association between oral cancer occurrence and Candida oral colonization and that the observed genotypic diversity of C. albicans strains may play a role in oral carcinogenesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Membrane complexes of Porphyromonas gingivalis were analyzed using two dimensional-blue native-PAGE. The molecular mass of the gingipain complexes, RgpA and Kgp, ranged from 450 kDa to greater than 1200 kDa, and did not change in single rgpA and kgp mutants indicating that the proteolytically processed polyproteins were independently capable of forming complexes. The outer membrane protein, LptO, which is essential for gingipain secretion, was found in up to seven different complex sizes. PG0026, also important for secretion, was observed to interact with the largest LptO complex [VII] at 480 kDa, supporting a cooperative role in secretion. Two pro-form RgpB intermediates formed a complex before cleavage of their C-terminal secretion signal domains (CTDs) such that complex formation may occur during secretion and processing. This may also be the case for other CTD-proteins as not only modified, mature RgpB, but also CPG70 was found to exist as multi-subunit complexes. RagA and RagB were observed in three different complex sizes. Elimination of the abundant gingipains enabled the identification of many inner and outer membrane protein complexes: TonB:ExbB:ExbD, Omp85, P51:PG2168, PorK:PorN, PG0056, PG0241, PG1430 and five proposed respiratory chain complexes (Mmd, Nqr, Rnf, Frd/Sdh and Atp)., Biological Significance: Porphyromonas gingivalis is a major oral pathogen associated with chronic periodontitis in humans. Secreted gingipains are considered major virulence factors of this pathogen and are secreted by a newly described type IX secretion system. This work has used 2D-BN-PAGE and MS to demonstrate that mature gingipains can independently form complexes and that substrate intermediates and mature secreted proteins of the type IX secretion system form multi-subunit complexes. Based on this work we propose that the substrates of this secretion system are secreted as large multi-subunit protein complexes. Two known important components of the secretion machinery, PG0026 and the integral outer membrane protein, LptO, were found to interact which would anchor PG0026 to the outer membrane and perhaps aid in the function of PG0026 to cleave the CTD from secreted substrates. The work has also identified more than 100 membrane proteins forming multi-subunit complexes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The development of smooth, featureless surfaces for biomedical microelectronics is a challenging feat. Other than the traditional electronic materials like silicon, few microelectronic circuits can be produced with conductive features without compromising the surface topography and/or biocompatibility. Diamond is fast becoming a highly sought after biomaterial for electrical stimulation, however, its inherent surface roughness introduced by the growth process limits its applications in electronic circuitry. In this study, we introduce a fabrication method for developing conductive features in an insulating diamond substrate whilst maintaining a planar topography. Using a combination of microwave plasma enhanced chemical vapour deposition, inductively coupled plasma reactive ion etching, secondary diamond growth and silicon wet-etching, we have produced a patterned substrate in which the surface roughness at the interface between the conducting and insulating diamond is approximately 3 nm. We also show that the patterned smooth topography is capable of neuronal cell adhesion and growth whilst restricting bacterial adhesion., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Recent research has examined associations between inflammation and mental health, and has increasingly focused on utilising younger samples to characterise the temporal relationship between inflammatory responses and the emergence of other symptoms. These studies have typically used blood to measure inflammation, although rates of detection for many inflammatory markers appear to be low. Saliva is a safe and low-cost alternative, and adult research has shown that levels of some salivary markers correlate well with those in serum. However, no research has examined this association in young people. This study examined 16 inflammatory markers in serum and saliva in 17 depressed adolescents and 18 healthy controls, aged 13-18 years. In general, detection rates were higher in saliva compared to in serum. When non-detectable levels were excluded, serum levels of C-reactive protein (CRP) correlated with salivary CRP (r=0.424, p=0.015), and this correlation appeared to only exist for those individuals with high levels of serum CRP (r=0.599, p=0.014). However, when non-detectable levels were included as zero, salivary levels of CRP, interleukin (IL)-2, IL-12p70, and interferon (IFN)-γ correlated with their serum counterparts. No significant clinical group differences in any acute phase proteins or cytokines were present. This study suggests that saliva can be used to measure inflammation in studies with adolescent participants, especially CRP, as it appears to correlate with systemic inflammation for those individuals who are expected to have high levels of inflammation. Implications for future directions in research on salivary inflammatory markers are discussed., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: To investigate and clarify physical and chemical properties of enamel affected by molar incisor hypomineralisation (MIH)., Methods: A series of in vitro studies were performed on extracted molars affected by MIH and sound teeth for controls. Tooth sections underwent Vickers microhardness testing before lapping and subsequent transverse microradiographic analysis and examination under polarised light microscopy. Carbonate content was determined by CO2 release from acid digestion. Unprepared and fractured surfaces were examined under scanning electron microscopy., Results: MIH-affected molars demonstrated a severe degree of hypomineralisation with an average mineral content of only 58.8%vol% mineral. Vickers microhardness was significantly reduced in MIH compared with controls (1.8±1.1 v 4.4±1.0 GPa, p<0.05) and polarised light microscopy revealed the bulk of MIH lesions had a porosity of ≤5% but also substantial areas of ≥10% and smaller areas exceeding 25% porosity. A surface layer was frequently observed on both intact and broken-down lesions and cervical regions of MIH teeth were typically spared. Carbonate content of MIH enamel was higher than control samples (6.6±2.1 v 4.4±1.1 wt%, p<0.05). Scanning electron microscopy showed that both the enamel rod and surface ultrastructure were defective. Clinical characteristics did not consistently correlate with all properties., Conclusions: The properties of MIH-affected enamel significantly differ from those of normal enamel and were highly variable, however some common characteristics were observed. Implications for aetiology and clinical management are discussed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Unlabelled: Caseinomacropeptide (CMP), the variably phosphorylated and glycosylated forms of the bovine milk protein fragment, κ-casein(106-169), is produced during cheese production and has been shown to have a range of antibacterial bioactivities., Objectives: To characterise the biofilm disruptive component of CMP and compare its activity with the known antimicrobial agents chlorhexidine and zinc ions., Methods: Streptococcus mutans biofilms were grown in flow cells with an artificial saliva medium containing sucrose and treated with CMP and the glycosylated forms of κ-casein(106-169) (κ-casein glycopeptide, KCG). The biofilms were imaged using confocal laser scanning microscopy (CLSM) and quantified by COMSTAT software analysis. A static biofilm assay and flow cytometric analysis were used to examine the mechanism of action of chlorhexidine and a combination of KCG with the known antimicrobial agent ZnCl2 (KCG-Zn)., Results: CLSM analysis showed that S. mutans produced robust, structured biofilms with an average thickness of 7.37μm and a biovolume of 3.88μm(3)/μm(2) substratum after 16h of incubation in the flow cell system. A single application of 10mg/mL CMP that contained 2.4mg/mL KCG significantly reduced total biofilm biovolume and average biofilm thickness by 53% and 61%, respectively. This was statistically the same as a 2.4mg/mL KCG treatment that reduced the total biovolume and average thickness by 59% and 69%, respectively, suggesting the KCG was the biofilm disruptive component of CMP. Chlorhexidine treatment (0.1%) caused similar effects in the flow cell model. KCG-Zn caused significantly more disruption of the biofilms than either KCG or ZnCl2 treatment alone. In a static biofilm model chlorhexidine was shown to work by disrupting bacterial membrane integrity whilst KCG-Zn had no effect on membrane integrity., Conclusions: KCG and KCG-Zn may have potential as natural biofilm disruptive agents., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A new glass-ionomer cement (GIC) (Fuji VII™ EP) includes 3% (w/w) casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) to enhance ion release., Objectives: To assess this new GIC compared with a GIC without CPP-ACP (Fuji VII™) with respect to ion release, changes in surface hardness and in mass under a variety of acidic and neutral conditions., Methods: Eighty blocks of Fuji VII™ (F7) and Fuji VII™ EP (F7EP) were subjected to three acidic solutions (lactic and citric acids pH 5.0, hydrochloric acid pH 2.0) and water (pH 6.9) over a three-day period. Ion release, surface hardness and weight measurements were carried out every 24h., Results: Higher calcium ion release from F7EP was observed under all acidic conditions. Increased inorganic phosphate ion release was observed for F7EP in hydrochloric and citric acids. Fluoride ion release was similar between F7 and F7EP under all conditions but was significantly higher in acids compared with water. After three days there was no significant difference in surface hardness (p>0.05) between the two materials under all conditions except hydrochloric acid. Minimal change in mass was observed for F7 and F7EP in water, lactic and hydrochloric acids, however citric acid caused significantly more mass loss compared with water (p<0.001)., Conclusion: Incorporation of 3% (w/w) CPP-ACP into F7 enhanced calcium and phosphate ion release, with no significant change in fluoride ion release and no adverse effects on surface hardness or change in mass., Clinical Significance Statement: GICs have the potential to release fluoride ions particularly under acidic conditions associated with dental caries and erosion. A new GIC containing CPP-ACP and fluoride releases not only fluoride ions but also calcium and phosphate ions under acidic conditions which should help to inhibit demineralisation associated with caries and erosion., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Soy beverages are water extracts of whole soybeans and are often promoted as a healthy alternative to bovine milk. Little analysis has been carried out to determine the effects of soy beverages on oral health, especially their potential acidogenicity., Objectives: The aim of this study was to determine the potential acidogenicity of a range of soy and bovine milk beverages., Methods: In vitro acid production by Streptococcus mutans was measured in soy and milk beverages at a constant pH of 6.5 or 5.5, as was the fall in pH over a 10 min period. The acid buffering capacity and calcium and phosphate concentrations (total and soluble) of the beverages were also determined., Results: The rate of acid production by S. mutans in the milk beverages was five to six times lower at pH 6.5 than in the soy beverages and three to five times lower at pH 5.5. Whilst the pH fall in the presence of S. mutans over 10 min was negligible in the milk beverages there was a significant decrease in pH in the soy beverages. This was also reflected in the lower buffering capacity of the soy beverages. The levels of soluble calcium in the soy beverages were lower than those in the milk beverages although total calcium contents were similar., Conclusions: Soy beverages have a higher potential acidogenicity than bovine milk beverages., Clinical Significance Statement: Patients consider soy beverages to be a healthy, low cariogenic alternative to other beverages, including bovine milk. This study shows that soy beverages have a higher potential acidogenicity than bovine milk and therefore may have a greater potential cariogenicity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Unlabelled: Dental products containing calcium phosphate and fluoride are claimed to enhance enamel remineralization over fluoride products., Objectives: To compare remineralization of enamel subsurface lesions by dental products with added calcium phosphate in a double-blind, randomized, cross-over in situ study., Methods: Human enamel specimens with subsurface lesions were prepared and inserted into intra-oral appliances worn by volunteers. A slurry (1g product plus 4 ml H(2)O) of each product was rinsed for 60s, 4 times per day for 10 days. Six products were tested (i) placebo, (ii) 1000 ppm F, (iii) 5000 ppm F, (iv) Tooth Mousse (TM), (v) TM plus 900 ppm F (TMP) and (vi) Clinpro with 950 ppm F. Calcium, inorganic phosphate and fluoride levels were measured in post-rinse/saliva samples using ion chromatography. Mineral content was measured using transverse microradiography., Results: Only TM and TMP significantly increased salivary calcium and phosphate levels. The products produced remineralization in the following order from lowest to highest: placebo<1000 ppm F=Clinpro<5000 ppm F

Introduction: White-spot lesions (WSL) might be susceptible to mechanical damage during orthodontic bracket and adhesive removal. The aims of this in-vitro study were to investigate enamel loss on bracket and adhesive removal when the brackets were surrounded by WSL and to determine the effect of remineralizing these lesions with a 1% (w/v) casein phosphopeptide amorphous calcium fluoride phosphate (CPP-ACFP) solution before bracket and adhesive removal., Methods: Precoated metal mandibular incisor brackets were centrally bonded onto polished third molars and WSL produced by exposure to a demineralization buffer for 4, 12, and 30 days (n = 20 per group). Half of the demineralized window was covered with acid-resistant nail varnish, and the specimens were then subjected to remineralization with 1% CPP-ACFP. Brackets and residual adhesive were removed, and enamel damage was assessed by digital photography, profilometry, and scanning electron microscopy. Lesion depth, mineral loss, and remineralization were measured by transverse microradiography., Results: WSL enamel around the bracket was more susceptible to iatrogenic damage at adhesive removal compared with sound enamel. Remineralization of lesions with 1% CPP-ACFP before adhesive removal significantly (P <0.002) reduced the area and depth of damage., Conclusions: Remineralizing WSLs with CPP-ACFP before adhesive removal reduced iatrogenic enamel damage., (Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

Objectives: Means of objectively assessing white spot enamel lesions (WSEL) are critical for determining their potential activity and monitoring the success of preventive treatments. The aim of this study was to determine whether surface pH measurements of WSEL changed during a preventive course of care designed to remineralize the lesions., Methods: Eight healthy subjects (1 male and 7 females) with at least one WSEL were recruited (19-64 years). Each subject was placed on a preventive treatment program including the daily application of a CPP-ACP paste (MI paste, GC Corp., Japan) with custom fitted trays for more than 6 months. The surface pH values of sound enamel and WSEL were monitored for up to 2 years using a micro-pH sensor. The visual appearance of the WSEL was monitored via digital photography, and images were analyzed qualitatively on a 5-point scale to assess the success of the remineralization preventive program. The relationship between the qualitative assessment of WSEL appearance and the WSEL pH was investigated using a Spearman's rho non-parametric correlation., Results: The surface pH of the WSEL was different to that of the sound enamel surrounding it in all patients at all times. All lesions showed visual improvement as the treatment period progressed. The pH of the WSEL increased towards that of sound enamel over the course of treatment significantly correlating with the visual improvement of the lesion (rho=0.63, p<0.0001)., Conclusions: The clinical assessment of WSEL surface pH changes with time may have utility as an additional objective measure for the assessment of WSEL activity., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Objectives: Chewing sugar-free gum has been shown to promote enamel remineralization. Manufacturers are now adding calcium to the gum in an approach to further promote enamel remineralization. The aim of this study was to compare the remineralization efficacy of four sugar-free chewing gums, two containing added calcium, utilizing a double-blind, randomized, crossover in situ model., Methods: The sugar-free gums were: Trident Xtra Care, Orbit Professional, Orbit and Extra. Ten subjects wore removable palatal appliances with four human-enamel half-slab insets containing subsurface demineralized lesions. For four times a day for 14 consecutive days subjects chewed one of the chewing gums for 20min. After each treatment the enamel slabs were removed, paired with their respective demineralized control slabs, embedded, sectioned and mineral level determined by microradiography. After 1-week rest the subjects chewed another of the four gums and this was repeated until each subject had used the four gum products., Results: Chewing with Trident Xtra Care resulted in significantly higher remineralization (20.67+/-1.05%) than chewing with Orbit Professional (12.43+/-0.64%), Orbit (9.27+/-0.59%) or Extra (9.32+/-0.35%). The form of added calcium in Trident Xtra Care was CPP-ACP and that in Orbit Professional calcium carbonate with added citric acid/citrate for increased calcium solubility., Conclusions: Although saliva analysis confirmed release of the citrate and calcium from the Orbit Professional gum the released calcium did not result in increased enamel remineralization over the normal sugar-free gums. These results highlight the importance of calcium ion bioavailability in the remineralization of enamel subsurface lesions in situ.

Objective: The aim of this study was to adapt a quantitative test for stimulated saliva, known as the Checkbuf test, to measure the buffering capacity of resting saliva and to evaluate its correlation with the Ericsson test for resting saliva., Study Design: Resting saliva was collected from 112 patients. Buffering capacity was assessed with the adapted Checkbuf test and 4 other methods: Ericsson test for resting saliva, Checkbuf test, and commercially available paper and liquid colorimetric tests for stimulated saliva. Data were analyzed by Cohen kappa coefficient to evaluate the agreement between the Ericsson test and the other tests., Results: There was a significant positive agreement between the Ericsson test for resting saliva and adapted Checkbuf test (kappa = 0.708). However, there were no agreements between the Ericsson test and the 3 other tests., Conclusions: This study demonstrated that the adapted Checkbuf test is useful for measuring resting saliva buffering capacity, even for subjects with low salivary flow rates.

Introduction: The demineralization of enamel adjacent to orthodontic brackets is a significant clinical problem. The purpose of this in-vitro study was to investigate the effect of sodium fluoride (Colgate Neutrafluor 9000 ppm) (NaF) and 10% casein phosphopeptide-amorphous calcium phosphate (GC Tooth Mousse) (TM) on enamel demineralization adjacent to orthodontic brackets., Methods: Forty specimens were sectioned from the buccal or lingual surfaces of extracted sound third molars. Twenty specimens had molar tubes bonded with composite resin (Transbond XT, 3M, St Paul, Minn) (CR), and 20 were bonded with resin-modified glass ionomer cement (Fuji Ortho LC, GC America, Alsip, Ill) (RMGIC). A 2-mm window for enamel demineralization was prepared. The specimens were randomly divided into 4 treatment groups: control, TM, TM/NaF (50/50 w/w), and NaF. The treatment solutions were placed around the bracket margins, and the specimens were immersed inverted into a carbopol demineralization solution at 37 degrees C. The enamel specimens were exposed for 96 hours, with the demineralization and topical solutions changed every 4 hours. Quantitative light-induced fluorescence (QLF) images were taken every 8 hours under controlled conditions. The difference in fluorescence (DeltaF) and the proportional DeltaF (%F) change between baseline and 96 hours was calculated., Results: RMGIC significantly reduced DeltaF and %F when compared with CR (ANOVA, P = .029 and P = .034, respectively). Application of TM with CR, NaF with CR, and TM/NaF with CR significantly reduced DeltaF and %F compared with the control CR (Tukey post-hoc test, P <.001). Application of TM/NaF with RMGIC significantly reduced DeltaF and %F compared with the control RMGIC (Tukey post-hoc test, P = .008, P = .019, respectively)., Conclusions: With the limitations of any in-vitro study, the following clinical conclusions can be drawn. The use of RMGIC alone can significantly decrease enamel demineralization compared with CR. The application of TM/NaF can provide significant additional prevention of enamel demineralization when RMGIC is used for bonding. The application of TM, NaF, or TM/NaF can significantly prevent enamel demineralization when CR is used for bonding. The use of both agents should be recommended for any at-risk orthodontic patient to provide preventive actions and potentially remineralize early (subclinical) enamel demineralization.

The salivary peptide histatin 5 has been reported to be an inhibitor of the Arg- and Lys-specific proteinases of Porphyromonas gingivalis, an oral pathogen associated with periodontitis. In this study a purified P. gingivalis proteinase preparation consisting of a complex of the Arg- and Lys-specific proteinases and adhesins was assayed using chromogenic substrates in the presence of histatin 5. Histatin 5 produced a concentration-dependent decrease in the initial rate of hydrolysis of the chromogenic substrates by both proteinases. However, pre-incubation of histatin 5 with the purified proteinase preparation or a P. gingivalis cell sonicate for 10 min prior to assay with the chromogenic substrates showed that under these conditions the salivary peptide did not decrease the initial rate of chromogen release. Mass spectrometric analysis revealed rapid degradation of histatin 5 at all four lysyl and all three arginyl residues by the P. gingivalis proteinases. This study demonstrates that histatin 5 is a substrate for the P. gingivalis extracellular Arg- and Lys-specific cysteine proteinases and not an inhibitor.

Multiphosphorylated segments of proteins are predicted to be in or near epitopes. However, due to the very hydrophilic nature of multiphosphorylated peptides, epitope mapping by ELISA using conventional microtitre plates can produce false negatives due to poor antigen adsorption. We have developed a sensitive ELISA for a multiphosphorylated peptide alpha(s1)-casein(59-79) containing five phosphoseryl residues using Nunc-Immuno Maxisorp modules for antigen adsorption. The peptide alpha(s1)-casein(59-79) was detected in the ELISA at antigen coating concentrations of 1.0 microg/ml and above, using rabbit anti-casein antibodies at a dilution of 1/10,000 in Tris-buffered saline containing 0.05% (w/v) Tween 20 and 1.0% (v/v) normal goat serum. At an antigen coating concentration of 10 microg/ml, anti-casein antibodies bound to alpha(s1)-casein(59-79) and produced an absorbance of more than 100 times background. Using conventional polyvinyl chloride and polystyrene plates the peptide alpha(s1)-casein(59-79) could only be detected at very high antigen coating concentrations of 1-10 mg/ml. The addition of 0.05% (w/v) Tween 20 to the blocking, antibody diluting and wash buffers of the ELISA was shown to significantly reduce nonspecific binding of the primary antibody. Further, the inclusion of normal goat serum in the blocking and antibody diluting buffers resulted in a small but significant increase in absorbance. The ELISA developed in this study has been used successfully with a range of enzymatically derived and synthetic peptides containing one to five phosphorylated residues such that it should have general applicability to the study of antigenicity of multiphosphorylated peptides.

The absence of supporting media in free solution high-performance capillary electrophoresis (HPCE) makes it an ideal system for the study of the relationship between electrophoretic mobility (mu(em)) and the molecular size and charge of proteins and peptides. In this review, the theory of electrophoresis, developed for rigid, insulating, spherical particles, is modified to develop models for the electrophoretic behaviour of proteins and peptides. For a given set of experimental conditions, mu(em) of a protein/peptide is proportional to its charge (q) and is inversely proportional to its Stoke's radius (r). Furthermore, mu(em) is most sensitive to changes in q and, as a consequence, the reliability of equations relating mu(em) to protein/peptide q and r is dependent upon the accurate calculation or determination of q. For convenience, q and r of proteins and peptides are generally expressed in terms of calculated valence (Zc) and molecular mass (M), respectively, both of which can be determined from the amino acid sequence of the protein/peptide. However, the calculation of q using Zc is made more complex by the effects of electrostatic charge suppression, such that Zc is an overestimation of actual charge. Charge suppression becomes increasingly significant as the protein/peptide charge increases, such that, for peptides, the relationship between q and Zc can be approximated by a logarithmic function. The mu(em) for peptides, therefore, can be approximated by the equation: mu(em) = ln(Zc + 1)/K Ms where s varies between 1/3 and 2/3, and K is a constant that is valid for a particular set of experimental conditions. The rather simplistic compensation for charge suppression in this equation is inadequate for proteins where the magnitude of charge suppression is greater and the mechanisms are more complex. For proteins, the relationship suggested for the prediction of mu(em) from Zc and M is: mu(em) = Zc/KFzMs where s again varies between 1/3 and 2/3 and Fz is a pH-independent proportionality factor defined as the quotient, Zc/Za, with Za being actual protein valence. The factor Fz can be determined empirically, however, it is valid only for the particular set of experimental conditions under which it is determined. For peptides, the mass exponent, s, approaches 1/3 when the peptides have high charge densities and open structures. However, s approaches 1/2 for peptides with lower charge densities that are capable of more randomized motion during electrophoresis. Finally, s approaches 2/3 for proteins, suggesting that the frictional forces acting on a protein undergoing electrophoretic motion are proportional to the surface area of these larger, more rigid, structures. In conclusion, the development of relationships between mu(em), M and Zc for peptides and proteins offers a powerful tool, not only for predicting electrophoretic mobility, but also for optimising HPCE separations, studying structural modifications (e.g. phosphorylation, glycosylation, deamidation, etc.), and for the investigation of surface charge characteristics and conformation.

Tryptic casein phosphopeptides containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu- have been shown to stablize amorphous calcium phosphate at neutral and alkaline pH and be anticariogenic in various in vitro, animal and human experiments. Furthermore, metal ion complexes of the casein phosphopeptides (CPPs) have potential as dietetic supplements to increase the bioavailability of calcium, iron, and other essential metal ions. In this study, we have used a Ca2+/ethanol selective precipitation procedure to produce a range of phosphopeptides from an alcalase digest of whole casein. The CPPs released by alcalase were truncated relative to those which are released by trypsin. The peptides could be grouped into those containing the cluster sequence as well as the group of tri-, di-, and monophosphorylated peptides. The two groups contained a number of homologous peptides of varying lengths resulting from the broad specificity of alcalase. Alcalase was observed to cleave peptide bonds on the carboxyl side of Glu, Met, Leu, Tyr, Lys, and Gln; however, of the twenty-six different cleavage sites, seventeen contained a Glu in the P1 position and of these, fifteen contained a hydrophobic residue in either the P2' or P3' positions. Furthermore, of the twenty-six cleavage sites identified, twenty-two contained a hydrophobic residue in either the P2' or P3' positions. Of the four other sites cleaved by alcalase, two contained a hydrophobic residue in the P1' position and one a hydrophobic residue in the P1 position.

Cysteine proteinases of Porphyromonas gingivalis have been implicated as major virulence factors in the development of periodontitis. Several groups have reported the characterisation of similar genes encoding the same arginine-specific thiol proteinase from P. gingivalis; however, the reported size and structure of the genes have varied. We report here the complete nucleotide sequence of the gene prtR that encodes a polyprotein containing the Arg-specific proteinase and multiple haemagglutinins/adhesins. The nascent polyprotein consists of a putative leader sequence and a prosequence followed by the 45 kDa Arg-specific proteinase and 44, 15, 17 and 27 kDa sequence-related adhesins in that order. The size and structure of the prtR are consistent with the size of the mRNA transcript (5.3 kb) and the size and sequences of the individual protein components purified from P. gingivalis.

We have purified from Porphyromonas gingivalis W50 a 45 kDa arginine-specific, thiol-activated, EDTA-sensitive endopeptidase, designated prtR. Oligonucleotide probes based on the N-terminal amino acid sequence were used to isolate a genomic fragment containing an open reading frame (3654 bp) with the potential to encode a 132 kDa protein including the prtR N-terminus. Analysis of this prtR gene revealed that the predicted nascent product contains a protease domain followed by a haemagglutinin domain and is post-translationally processed by proteolytic (possibly autolytic) events to produce a 43-54 kDa arginine-specific, thiol protease and a 41-53 kDa haemagglutinin. Comparison of the prtR with the P. gingivalis prtH gene suggests that the prtH gene product also contains protease and haemagglutinin domains but in the reverse order to that in the prtR. An overlapping but shifted reading frame at the 3' end of the prtR encodes the 5' region of the prtH.

The heat-stable phosphocarrier protein (HPr) of Streptococcus mutans was extracted from whole cells using sodium lauroylsarcosinate/EDTA and purified to homogeneity by a single-step, ion-exchange chromatographic procedure. The complete amino acid sequence of the protein was determined from peptides generated by trypsin, alpha-chymotrypsin, endoproteinase Glu-C, and cyanogen bromide treatment. The HPr from S. mutans contains 86 or 87 amino acyl residues, depending on removal of the N-terminal Met and the protein shows high sequence homology with HPr from other Gram-positive bacteria. The predicted tertiary structure of the S. mutans HPr, from model building by homology, is an open-faced beta-sandwich consisting of two alpha-helices and a four-stranded antiparallel beta-sheet.

Multiple phosphoseryl-containing sequences of proteins stabilize amorphous calcium phosphate and have been implicated in the regulation of biomineralization, protein structure, and enzyme activity. To facilitate studies on the identification and characterization of multiple phosphoseryl-containing sequences of proteins we have developed a simple and efficient purification procedure involving precipitation of Ca2+/ethanol-induced aggregates of the multiple phosphoseryl-containing peptides from enzymic digests. The multiple phosphoseryl-containing peptides of a tryptic digest of casein were selectively precipitated using Ca2+ (20 mol/mol protein) and 50% (v/v) ethanol at pH 3.5, 4.6, and 8.0. The individual peptides of the precipitates were purified using anion-exchange fast-performance liquid chromatography and reversed-phase HPLC and then identified by solid-phase sequence analysis and amino acid composition analysis after vapor-phase hydrolysis. Prior to sequence analysis the phosphopeptides were covalently coupled to arylamine membranes and the phosphoseryl residues converted to S-ethylcysteinyl residues by calcium-ion-catalyzed beta-elimination in the presence of ethanethiol. The modified peptides were sequenced using an Applied Biosystems Inc. automated protein sequencer fitted with a membrane cartridge. Only peptides containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)- were precipitated by Ca2+/ethanol at pH 3.5. The pH 4.6 precipitate contained all the cluster peptides plus two diphosphorylated peptides containing -Ser(P)-Glu-Ser(P)- and -Ser(P)-Thr-Ser(P)-. At pH 8.0, a monophosphorylated peptide containing -Ser(P)-Glu-Glu- was also present in the precipitate with the diphosphorylated and cluster peptides. The recoveries of the peptides in the pH 8.0 selective precipitate ranged from 83 to 95% of that present in the hydrolysate.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple phosphoseryl-containing sequences of peptides and proteins stabilize amorphous calcium phosphate at neutral and alkaline pH and have been implicated in the nucleation/regulation of biomineralization. In an approach to analyze these peptides using capillary zone electrophoresis (CZE) we have attempted to relate the absolute electrophoretic mobility of various casein phosphopeptides to their physicochemical properties. Multiple phosphoseryl-containing peptides were selectively precipitated from enzymic digests of sodium caseinate and further purified using RP-HPLC and anion-exchange fast protein liquid chromatography. Purified fractions were then analyzed by CZE. Absolute electrophoretic mobilities of 13 peptides were determined by measurement of migration times relative to that of a neutral marker, mesityl oxide. A linear relationship (r2 = 0.993) was obtained between absolute electrophoretic mobility and q/M(r)2/3 where q is the net negative charge of the peptide calculated using relevant pKa values and M(r) is the molecular mass. M(r)2/3 is a measure of the surface area of a sphere that has a volume proportional to the M(r) of the peptide and relates to the frictional drag exerted on the peptide during electrophoretic migration. As absolute electrophoretic mobility is influenced by charge and size CZE can be used to monitor peptide phosphorylation, dephosphorylation, deamidation and truncation. This technique therefore would be suitable for quantitative analysis of peptide substrates in kinase and phosphatase studies. In conclusion CZE is a rapid and efficient technique for the resolution of multiple phosphoseryl-containing peptides from enzymic digests of casein.

This study examined the influence of acetylsalicylic acid an inhibitor of prostaglandin synthesis, on orthodontic tooth movement induced with light spring forces in the guinea pig. The animal model was shown to permit reliable and accurate recording of tooth movement up to 28 days. Tooth movement was found to be highly correlated with spring forces, indicating that the model provided a sensitive test of the effect of aspirin on tooth movement. Aspirin was administered orally at the rate of 65 mg/kg per day in three divided doses and was found to effectively inhibit prostaglandin synthesis at the level of the bronchioles. However, aspirin did not appear to significantly affect tooth movement. Thus prostaglandins may not be the only mediators of the bone resorption associated with tooth movement induced by light orthodontic forces under these experimental conditions.