Native and chimeric metal-binding lactate dehydrogenase as detection and protection tools for oxidative stress induced by Fenton's reaction

In the present study, a simple and reliable antioxidant screening technique based on lactate dehydrogenase (LDH) oxidation by Cu2+-mediated Fenton’s reaction has successfully been developed. Oxidation of LDH by hydroxyl radical consequently leads to enzymatic inactiva-tion, while addition of antioxidants can protect and regain enzyme activity. This method dem-onstrated a high feasibility on detecting of antioxidative activity of lipophilic (e. g. alpha-Tocopherol and beta-Carotene) and hydrophilic compounds (e. g. glutathione, mannitol and thiourea) in a single assay. Results from linear correlation curves revealed that the IC50 were in the order of beta-carotene (3.45 µg/ml) > alpha-Tocopherol (52.31 µg/ml) > Mn(II)-bacitracin (109.37 µg/ml) > glutathione (122.63 µg/ml). Detailed investigations revealed that oxidation of LDH resulted in enzyme degradation, which was metal- and time-dependent mechanism. Therefore, further experiments were conducted to determine whether extension of the N-terminus of LDH with metal-binding regions possesses protective effect against the inactivation process. Genetic construction of chimeric LDH carrying two and four repetitive se-quences of cadmium binding peptide (CdBP), designated as CdBP2LDH and CdBP4LDH, has been carried out. From our findings, the CdBP2LDH and the CdBP4LDH exhibited protective action and enzyme activity regained 20-30 % and 70 % higher than that of the native LDH, respectively. Two possible mechanisms have been proposed to play important role in protec-tion against metal-mediated Fenton’s reaction: i) changing in redox potential of Cu2+ in metal-peptide complex, and ii) taking away of Cu2+ ion from the crucial amino acids by metal satu-ration at the cadmium-binding peptides

A Model for the Analysis of Caries Occurrence in Primary Molar Tooth Surfaces

Recently methods of caries quantification in the primary dentition have moved away from summary ‘whole mouth’ measures at the individual level to methods based on generalised linear modelling (GLM) approaches or survival analysis approaches. However, GLM approaches based on logistic transformation fail to take into account the time-dependent process of tooth/surface survival to caries. There may also be practical difficulties associated with casting parametric survival-based approaches in a complex multilevel hierarchy and the selection of an optimal survival distribution, while non-parametric survival methods are not generally suitable for the assessment of supplementary information recorded on study participants. In the current investigation, a hybrid semi-parametric approach comprising elements of survival-based and GLM methodologies suitable for modelling of caries occurrence within fixed time periods is assessed, using an illustrative multilevel data set of caries occurrence in primary molars from a cohort study, with clustering of data assumed to occur at surface and tooth levels. Inferences of parameter significance were found to be consistent with previous parametric survival-based analyses of the same data set, with gender, socio-economic status, fluoridation status, tooth location, surface type and fluoridation status-surface type interaction significantly associated with caries occurrence. The appropriateness of the hierarchical structure facilitated by the hybrid approach was also confirmed. Hence the hybrid approach is proposed as a more appropriate alternative to primary caries modelling than non-parametric survival methods or other GLM-based models, and as a practical alternative to more rigorous survival-based methods unlikely to be fully accessible to most researchers

Hypomineralized Second Primary Molars as Predictor of Molar Incisor Hypomineralization

Molar incisor hypomineralization (MIH) is a developmental defect of dental enamel that shares features with hypomineralized second primary molars (HSPM). Prior to permanent tooth eruption, second primary molars could have predictive value for permanent molar and incisor hypomineralization. To assess this possible relationship, a cross-sectional study was conducted in a sample of 414 children aged 8 and 9 years from the INMA cohort in Valencia (Spain). A calibrated examiner (linear-weighted Kappa 0.83) performed the intraoral examinations at the University of Valencia between November 2013 and 2014, applying the diagnostic criteria for MIH and HSPM adopted by the European Academy of Paediatric Dentistry. 100 children (24.2%) presented MIH and 60 (14.5%) presented HSPM. Cooccurrence of the two defects was observed in 11.1% of the children examined. The positive predictive value was 76.7% (63.9-86.6) and the negative predictive value 84.7% (80.6-88.3). The positive likelihood ratio (S/1-E) was 10.3 (5.9-17.9) and the negative likelihood ratio (1-S/E) 0.57 (0.47-0.68). The odds ratio was 18.2 (9.39-35.48). It was concluded that while the presence of HSPM can be considered a predictor of MIH, indicating the need for monitoring and control, the absence of this defect in primary dentition does not rule out the appearance of MIH

The Microfloral Analysis of Secondary Caries Biofilm around Class I and Class II Composite and Amalgam Fillings

<p>Abstract</p> <p>Background</p> <p>Secondary caries is responsible for 60 percent of all replacement restorations in the typical dental practice. The diversity of the bacterial sources and the different types of filling materials could play a role in secondary caries. The aim of this study was to determine and compare the microbial spectrum of secondary caries biofilms around amalgam and composite resin restorations.</p> <p>Methods</p> <p>Clinical samples were collected from freshly extracted teeth diagnosed with clinical secondary caries. Samples were categorized into four groups according to the types of restoration materials and the classification of the cavity. Biofilms were harvested from the tooth-restoration interface using a dental explorer and after dilution were incubated on special agars. The bacteria were identified using the biochemical appraisal system. Statistical calculations were carried out using SPSS11.5 software to analyze the prevalence of the bacteria involved in secondary caries.</p> <p>Results</p> <p>Samples from a total of four groups were collected: two groups were collected from amalgam restorations, each had 21 samples from both Class I and Class II caries; and the other two groups were from composite resin restorations, each had 13 samples from both class I and class II caries. Our results showed: (1) Anaerobic species were dominant in both restoration materials. (2) In terms of the types of individual bacteria, no significant differences were found among the four groups according to the geometric mean of the detected bacteria (P > 0.05). However, there were significant differences among the detected bacteria within each group (P < 0.05). The composition of each bacterium had no statistical difference among the four groups (P > 0.05), but showed significant differences among the detected bacteria in each group (P < 0.05). (3) Among the four groups, there were no significant differences for the detection rate of each bacterium (P > 0.05), however, the detection rate of each bacterium within each group was statistically different among the detected bacteria (P < 0.05).</p> <p>Conclusions</p> <p>The proportion of obligatory anaerobic species was much greater than the facultative anaerobic species in the biofilm of secondary caries. Statistically, the materials of restoration and the location of secondary caries did not show any significant effects on the composition of the microflora.</p

The influence of cadmium stress on the content of mineral nutrients and metal-binding proteins in arabidopsis halleri

We investigated the influence of cadmium stress on zinc hyperaccumulation, mineral nutrient uptake, and the content of metal-binding proteins in Arabidopsis halleri. The experiments were carried out using plants subjected to long-term cadmium exposure (40 days) in the concentrations of 45 and 225 μM Cd2+. Inductively coupled plasma-mass spectrometry, size exclusion chromatography coupled with plasma-mass spectrometry, and laser ablation inductively coupled plasma-mass spectrometry used for ablation of polyacylamide gels were employed to assess the content of investigated elements in plants as well as to identify metal-binding proteins. We found that A. halleri is able to translocate cadmium to the aerial parts in high amounts (translocation index >1). We showed that Zn content in plants decreased significantly with the increase of cadmium content in the growth medium. Different positive and negative correlations between Cd content and mineral nutrients were evidenced by our study. We identified more than ten low-molecular-weight (<100 kDa) Cd-binding proteins in Cd-treated plants. These proteins are unlikely to be phytochelatins or metallothioneins. We hypothesize that low-molecular-weight Cd-binding proteins can be involved in cadmium resistance in A. halleri