Sergio Line

During development, oxidative stress is hypothesized to mediate embryotoxicity, which may be intensified by exposition to environmental factors and by genetic variations in the enzymes involved in protecting cells from these damaging effects, including superoxide dismutase (SOD) and paraoxonase (PON). The aim of this study was to evaluate the influence of single-nucleotide polymorphisms (SNP) in genes associated with the neutralization of oxidative stress (SOD and PON family members) in the risk of nonsyndromic oral cleft in the Brazilian population. Initially, we tested for association between 28 SNP in SOD1, SOD2, SOD3, PON1, PON2, and PON3 among 325 nonsyndromic cleft lip with or without cleft palate (NSCL±P) case-parent trios. Multiple logistic regression analyses were used to explore gene, GxG and GxE, involving factors that induce oxidative stress accumulation during pregnancy. Signals still significant after both Bonferroni correction and in permutation test were subsequently...

A method for isolation of the neurite outgrowth promoting fragment of mouse laminin (fragment 8) is described in this paper. Besides producing excellent yields, this method was shown to be fast and practical, since it is based on a single step which consists in an ion exchange chromatography of elastase digested laminin.

Polymorphisms, such as a guanine inserted at position -1607 in the promoter region of human matrix metalloprotenase 1 (MMP-1) or a C-1562T substitution in the MMP-9 gene, have been shown to increase the transcriptional activity of these MMPs. The objective of this study was to investigate the possible relationship between these polymorphisms and early implant failure. Genomic DNA from oral mucosa was amplified by polymerase chain reactions (PCRs) and analyzed by restriction endonucleases. The significance of the differences in observed frequencies of polymorphisms was assessed by the chi-square and Fisher exact tests. The test group comprised patients with early failure of osseointegrated oral implants. In the MMP-1 gene, 2G allele was observed in 25% of the control group and in 50% of the test group (P = .013). The genotype 1G/1G was found in 61.5% of the control group, while all patients in the test group had the genotype 1G/2G (P < .001). No differences were seen in the allele and genotype frequencies in the MMP-9 gene among the groups (P = .15 and P = .13, respectively). These results suggest that polymorphism in the promoter region of the MMP-1 gene may be associated with early implant failure, while polymorphism in the promoter region of the MMP-9 gene may not have a relationship with implant loss.

Temporomandibular joint disorders (TMJD) affect women with greater frequency than men, and sex hormones may contribute to this female predominance. Therefore, this study investigated whether estrogen receptor-α (XbaI/PvuII) single nucleotide polymorphisms (SNPs) are associated with TMJD in women. DNA was obtained from 200 women with TMJD (100 with chronic pain and 100 with signs of TMJD but no pain) diagnosed according to the Research Diagnostic Criteria for Temporomandibular Disorder (RDC/TMD) and 100 control women without TMJD. Restriction fragment length polymorphisms of polymerase chain reaction products were used to analyze XbaI and PvuII SNPs in DNA fragments. A model directly characterizing specific DNA sequence variants based on the risk haplotypic structure implemented with the EM algorithm was used to analyze the data. The [GC] haplotype of the XbaI locus was significantly more prevalent in both TMJD groups when compared with the control group (P = .0012). Specifically, the [GC] haplotype was more prevalent within the painful TMJD group versus the control group (OR = 3.203, 95% CI = 1.633, 6.284) and in the TMJD no pain versus the control group (OR = 2.51, 95% CI = 1.267, 4.97). In conclusion, the presence of [GC] haplotype in the XbaI locus may increase the susceptibility of women to develop TMJD.This study suggests that a polymorphism in the estrogen receptor may increase the risk of women developing temporomandibular joint disorder. This finding may elucidate the interindividual differences in the contribution of estrogen to TMJD, the genetic influences on TMJD predisposition, and may serve as the basis for future treatment tailoring, which could enhance outcomes for these patients.

Page 1. Page 2. LINE AND BERGQVIST-ENAMEL OF ITABORAI MAMMALS 925 TABLE 1. First lower molar width (mm) of Itaboraf ungulates. Ab-breviations: N, number of specimens; SD, standard deviation; =, trans-verse HSB; 11, vertical HSB. Taxa N Mean (mm) SD HSB ...

The mapping of the field of influence of specific regulatory molecules can provide a great deal of information on the molecular strategies that underlie the changes in the developmental program and macroevolutionary process. The strategy in this study was to use the variation in the number of teeth in the affected individuals of three mutant families with hypodontia, to determine the relative influence (relative molecular morphogenetic field) of MSX 1 and PAX 9 genes on the dental field. The variations in the pattern of symmetry of tooth agenesis were used in order to estimate the developmental stability of these genes. The approach used in the present work can help to explore new hypotheses linking development with the patterning of dentition during mammalian evolution. Furthermore, the developmental changes can be linked to changes in the molecular morphogenetic field of specific genes.

Chronic periodontitis (CP) is a chronic inflammatory disease independently associated with higher incidence of oral cavity squamous cell carcinoma (OSCC). However, the molecular mechanism responsible for this increased incidence is unknown. Here we profiled the DNA methylome of CP patients and healthy controls and compared to a large set of OSCC samples from TCGA. We observed a significant overlap between the altered DNA methylation patterns in CP and in OSCC, suggesting an emergence of a pre-neoplastic epigenome in CP. Remarkably, the hypermethylated CpGs in CP were significantly enriched for enhancer elements. This aberrant enhancer methylation is functional and able to disrupt enhancer activity by preventing the binding of chromatin looping factors. This study provides new insights on the molecular mechanisms linking chronic inflammation and tumor predisposition, highlighting the role of epigenetic disruption of transcriptional enhancers.

Tooth germ development is associated with morphological and biochemical changes of the dental papilla and enamel organ. Enzymes with gelatinolytic activities were studied by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzymography in tooth germ of newborn to 15-day-old rats. Three major bands with gelatinolytic activity were detected at all periods and characterized as the latent and active forms of MMP-2 using their molecular weight and activity dependent on Zn++ and Ca++ ions as criteria. Expression and activity of MMP-2 increased progressively from 0 to 15 days after birth. Mechanical separation of the tooth germ from 10-day-old rats showed that the gelatinolytic activity was localized mainly in the dental papilla and not the dental organ. These data indicate that the expression and activity of MMP-2 varies during the development and maturation of rat first molar tooth germ.

The aim of the present work was to investigate birefringence and morphology of the secretory-stage enamel organic extracellular matrix (EOECM), and structural and mechanical properties of mature enamel of upper incisors from adult rats that had been treated with pamidronate disodium (0.5 mg/kg/week for 56 days), using transmitted polarizing and bright-field light microscopies (TPLM and BFLM), energy-dispersive X-ray (EDX) analysis, scanning electron microscopy (SEM) and microhardness testing. BFLM showed no morphological changes of the EOECM in pamidronate and control groups, but TPLM revealed a statistically significant reduction in optical retardation values of birefringence brightness of pamidronate-treated rats when compared with control animals (p0.05). The present study indicates that pamidronate can affect birefringence of the secretory-stage EOECM, which does not seem to be associated with significant changes in morphological and/or mechanical properties of mature enamel.

A polymorphism in the promoter region of the transforming growth factor beta-1 (TGF-beta1) gene was described at position -509. This polymorphism represents a C-to-T base exchange, which creates a YY1 consensus sequence in an area involved with down transcription regulation. This polymorphism has been associated with risk for asthma and allergies. In this study we investigated the association between this polymorphism and chronic periodontitis severity. Genomic DNA from oral mucosa of 87 Caucasian subjects was amplified by PCR, and digested with Eco81I restriction endonuclease. The alleles were separated by polyacrylamide gel electrophoresis. The differences in genotype distribution from those expected by Hardy-Weinberg equilibrium, and the significance of the differences in observed frequencies of the polymorphism in moderate and severe disease and healthy groups were assessed by the chi2 test. There was a difference in the presence of the different alleles and genotypes among the healthy, moderate and severe periodontitis groups. The allele T was seen at 57.7% in the group with severe periodontitis and 37.8% and 35.4% in the healthy group and moderate periodontitis group, respectively (p=0.0387). The genotype T/T was found at 38.5% in the group with severe periodontitis, and at a frequency of 8% in the healthy group (p=0.0258). These results demonstrate that the polymorphism at bp -509 in the TGF-beta1 promoter may have a small effect on the modulation of the inflammatory process during periodontitis.

Tumor necrosis factor-alpha (TNF-alpha) is a potent inflammatory mediator with bone resorption activity. Polymorphisms in the promoter region of the human TNF-alpha gene have been shown to affect the levels of this cytokine and have been associated with a variety of diseases. The aim of this study was to investigate the possible relationship between early implant failure and a single nucleotide polymorphism (SNP) in the -308 promoter region of the TNF-alpha gene. A sample of 66 nonsmokers was divided into 2 groups: a test group comprising 28 patients (mean age, 52.7 years) with one or more early failed implants and a control group consisting of 38 individuals (mean age, 43.2 years) with one or more healthy implants. Genomic DNA from buccal mucosa was amplified by the polymerase chain reaction (PCR), analyzed by restriction fragment length polymorphism (RFLP), and submitted to polyacrylamide gel electrophoresis to distinguish allele G and allele A of the TNF-alpha (-308) gene polymorphism. Differences in the allele and genotype frequencies between control and test groups were assessed by chi-squared test (P <0.05). No significant difference was observed in the allele (P = 0.4635) and genotype (P = 0.4445) distribution of the polymorphism when control and failure groups were compared. The results indicate that the TNF-alpha (G-308A) gene polymorphism is not associated with early implant failure, suggesting that its presence alone does not constitute a genetic risk factor for implant loss in the Brazilian population.

... Recebimento: 07/11/02 Aceite: 02/12/02 Correspondence to: Maria Cristina Leme Godoy dosSantos Faculdade de Odontologia de Piracicaba-UNICAMP Av. Limeira 901. ... 10. McKinney RV Jr, Steflick DE, Koth DL, Singh BB. The scientific basis for dental implant therapy. ...

1. Fragments P1 and E8, the result of two different enzymatic digestions of the laminin molecule, represent interaction sites of laminin with specific cell receptors. By using negative and positive affinity purification of a rabbit antiserum against mouse laminin we have generated antibodies to these two fragments. 2. Antibodies against P1 were able to immunoprecipitate fragment E8 from elastase-digested laminin. By liquid phase competition experiments we demonstrated that the epitopes shared by P1 and E8 are a minor portion of the antigenic determinants of P1. When we checked for the presence of these shared epitopes in the human laminin molecule, they were the major fraction of the interspecies antigenic conservation. 3. A similar approach using polyclonal antibodies against human laminin has confirmed these results. 4. The shared epitopes present in both mouse and human laminin molecules seem to be spatially determined, because antibodies against these sites did not bind to fully denatured laminin. 5. Since human and mouse laminin bind to cell receptors and to other extracellular matrix proteins from both species, we conclude that these antigenic determinants may represent the actual sites for at least some of these interactions.

To explore the genetic relationship between anti-laminin and anti-DNA autoantibodies (autoAb), VH gene and gene family expression were determined among autoAb derived from an individual 6-mo-old MRL-lpr/lpr mouse. Whereas 85% of the anti-DNA Ig were identified by one of two VH family probes, 7183 and VHJ558, none of the anti-laminin antibodies (Ab) examined were recognized by these probes. Subsequent V region sequence analysis of three of the anti-laminin Ab revealed that they in fact utilized a J558 VH gene (VH50). Furthermore, FR2 and CDR2 oligonucleotide probes complementary to VH50 recognized multiple anti-laminin Ab by Northern blot analysis; the FR2 probe recognized two control anti-DNA Ab, but neither probe recognized anti-DNA Ab from the same mouse. Polymerase chain reaction amplification of MRL-lpr/lpr genomic liver DNA using primers generated from VH50 and Vk50 sequences indicated that all three anti-laminin Ig have a single replacement mutation in both their VH and Vk genes. Search of the nucleic acid databases revealed that both germline VH and Vk genes are expressed unmutated by murine lupus anti-dsDNA autoAb, previously sequenced in other laboratories. Sequence comparisons suggest that differences in anti-DNA and anti-laminin reactivity may be dependent upon somatically generated differences in the CDR3 regions of the H and L chains. The results indicate that lupus anti-laminin Ab can arise from distinct B cell populations but express the same unmutated germline V region genes as lupus anti-dsDNA autoAb. They further raise the possibility that these distinct B cell populations may be activated and expanded either: independently, by distinct Ig receptor ligands such as the Ag, laminin and DNA; or simultaneously, by a common ligand such as an anti-Id recognizing a common V region epitope.

Two polymorphisms in the promoter region of human MMP-1 gene, an insertion of a guanine at position -1607 and A-519G substitution, have been shown to increase the transcriptional activity of these matrix metalloproteinases (MMPs). The objective of this study was to investigate the possible relationship between these polymorphisms and early implant failure. A sample of 104 nonsmokers was divided into 2 groups: a test group comprising 44 patients with 1 or more early failed implants and a control group consisting of 60 individuals with 1 or more healthy implants. Genomic DNA from oral mucosa was amplified by polymerase chain reaction and analyzed by restriction endonucleases. The significance of the differences in observed frequencies of polymorphisms were assessed by chi2 tests. The G-1607GG polymorphism with the genotype G/G was observed at a frequency of 62% in the control group, while in the test group this genotype was noted in 34% of the individuals (P = .011). The allele G was found at a frequency of 75% in control group and 61.66% in the test group (P = .05). No significant differences were seen in the genotypes and allele frequencies in the A-519G polymorphism among the groups (P = .064 and P = .124, respectively). The distribution of the haplotypes arranged as alleles and genotypes showed a significant difference between control and test groups (P = .031 and P = .002, respectively). On the basis of this study of 60 patients who experienced no implant failure and 44 patients who experienced implant failure, the results suggest that G-1607GG polymorphism in MMP-1 gene is associated with early implant failure, while A-519G polymorphism in MMP-1 gene does not show a significant relationship with implant loss. This study also suggests that haplotypes G-1607GG and A-519G of MMP-1 may be associated with the osseointegration process.

Transforming growth factor-beta 1 is a multifunctional cytokine involved in extracellular matrix deposition, reduction of inflammation, and promotion of wound healing. Single nucleotide polymorphisms in the promoter region of human transforming growth factor-beta 1 gene, C-509T and G-800A, have been shown to increase the transcriptional activity of this cytokine and have been associated with a variety of diseases. The objective of this study was to investigate the possible association between these single nucleotide polymorphisms and the early implant failure. A sample of 68 nonsmoking patients was divided into two groups: a test group comprising 28 patients with one or more early failed implants and a control group consisting of 40 individuals with one or more healthy implants. Genomic DNA from oral mucosa was amplified by polymerase chain reaction and analyzed by restriction fragment length polymorphism. The significance of the differences in observed frequencies of single nucleotide polymorphisms was assessed using the chi square test and Fisher's exact test. The cited single nucleotide polymorphisms in transforming growth factor-beta 1 were analyzed in combination as haplotype using the computer program ARLEQUIN. The authors did not observe significant differences in the allele and genotypes to both single nucleotide polymorphisms of transforming growth factor-beta 1 gene (C-509T and G-800A) between control and early implant failure groups. The distribution of the haplotypes arranged as allele and genotypes were similar between control and test groups. These results indicate that C-509T and G-800A polymorphisms in the transforming growth factor-beta 1 gene are not associated separately or in haplotype combinations with early implant failure, suggesting that the presence of those single nucleotide polymorphisms alone do not constitute a genetic risk factor for early implant failure in the Brazilian population.

To determine the bond strengths promoted by an adhesive system to human, bovine, and porcine enamel and dentin, and compare their etched micromorphology by scanning electron microscopy. Thirty sound freshly extracted teeth were used in this study: ten human third molars, ten bovine incisors, and ten porcine molars. The crowns of human (H), bovine (B), and porcine (P) teeth were ground with 600-grit SiC paper to expose either enamel (E) or mid-depth dentin (D) surfaces. After application of the adhesive resin, composite crowns approximately 8 mm high were built up with TPH Spectrum composite. After 24 h of water storage, specimens were serially sectioned in the buccal-lingual direction to obtain 0.8 mm slabs, which were trimmed to an hourglass shape of approximately 0.8 mm2 at the bonded interface. Specimens were tested in tension in a universal testing machine (0.5 mm/min). Results were statistically analyzed with ANOVA and Tukey's test at the 95% confidence level. Tukey's test showed significant differences between bond strengths obtained on enamel and dentin (p < 0.05). However, there were no statistically significant differences in microTBS between human, bovine, and porcine teeth. SEM observations revealed a similar dentinal morphology for the three species. However, porcine enamel specimens presented a very different distribution of enamel prisms. Bovine teeth proved to be possible substitutes for human teeth in either dentin or enamel bond testing. However, even though porcine teeth provided enamel and dentin bond strengths similar to human and bovine teeth, enamel morphology presented a very different configuration.

Objective: Aging is a complex process which includes telomere shortening, DNA and cells components oxidative stress, mutation accumulation, apoptosis, changes in cell renewal. Recently, epigenetic modifications have been associated with this process, such as DNA methylation patterns that change with increasing age and contribute to age related disease. This change can affect the expression of genes related to chromatin remodeling. EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that targets lysine-27 of histone H3. The methylation in the H3-K27 is a chromatin mark that is commonly associated with silencing of genes. Studies on human normal senescent cells show that EZH2 is frequently down-regulated. The aim of this study was to investigate whether methylation pattern in the promoter region of EZH2 gene changes with aging, since this promoter shows CpG islands which can become methylated, leading the epigene...

Single nucleotide polymorphisms in the promoter region of the human interleukin (IL)-2 (T-330G) and IL-6 (G-174C) genes have modified the transcriptional activity of these cytokines and are associated with several diseases. The aim of this study was to investigate the possible relationship between these single nucleotide polymorphisms and early implant failure. A sample of 74 nonsmokers was divided into 2 groups: test group comprising 34 patients (mean age 49.3 years) with >or=1 implants that failed and control group consisting of 40 patients (mean age 43.8 years) with >or=1 healthy implants. Genomic deoxyribonucleic acid from oral mucosa was amplified by polymerase chain reaction and analyzed by restriction fragment length polymorphism. Monte Carlo simulations (P < 0.05) were used to assess differences in allele and genotypes frequencies of the single nucleotide polymorphisms between the 2 groups. No significant differences were observed in the allele and genotypes distrib...

Objective: The purpose of this study was to evaluate the gelatinases activity in undecalcified dentin from human molars crowns by in situ zymography. Method: Intact third molars were extracted and slices (thickness: 0.5mm) from dentin crown were obtained. The slices were polishing using silicon carbide papers and washed in deionized water. The samples were incubated with or without DQ gelatin in 50 mM TrisCaCl2 for 2 hours at 37 °C, and then analyzed under confocal microscopy. Result: The results showed that it was possible detected gelatinolytc activity in whole crown dentin. High activity of the gelatinases was observed in the odontoblastics process and in the predentin. Conclusion: It was possible to evidence gelatinases activity in undecalcified dentin from human molars, and it activity was not uniformly distributed over different compartments of the dentin. In situ zymography could be a tool in studies investigating the role of the gelatinases in the dentin development and degr...

Objective: The aim of this study was to assess whether chronic periodontitis and P. gingivalis LPS can modulate gene expression of the some regulatory factors of epigenetic events. Method: Total RNA was extracted from biopsies of gingival tissue from sites with or without periodontitis, and after cDNA synthesis gene expression of DNMT1 (DNA methyltransferase 1), DNAMT3a (DNA methyltransferase 3a), histone demethylases JMJD3 and UTX were evaluated by qRT-PCR. Primary gingival fibroblast culture and keratinocytes (HaCaT) were treated with medium containing P. gingivalis LPS or P. gingivalis LPS vehicle for 24hs, and then cell viability were assessed by MTT test, total RNA were extracted and after cDNA synthesis gene expression of DNMT1,DNMT3a, JMJD3 and UTX were evaluated by qRT-PCR. Result: No significant differences were found in the gene expression analysis between healthy gingival tissues and gingival tissue from pacients with periodontitis. The results showed that LPS downregulat...

Objective: SOCS-1 is a member of suppressor of cytokine signaling (SOCS) proteins that inhibits cytokine signaling function via JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription) and has been shown to play an important role in regulating inflammation and carcinogenesis. Epigenetic events such as DNA methylation may control gene expression, including SOCS-1. The CpG islands hypermethylation was related to chronic inflammation and the hypermethylation of SOCS-1 gene promoter has been observed in different epithelial tumors. Thus, the aim of this study was to investigate the DNA methylation status on the SOCS-1 gene promoter in chronic periodontitis. Method: Eighty DNA samples were purified from gingival tissue (40 healthy individuals (control group) and 40 individuals with chronic periodontitis (periodontitis group). After, DNA was treated with sodium bisulfate to convert unmethylated cytosine in timine, not affecting methylated cytosine. The analysis o...

Objective: Matrix metalloproteinase-3 (MMP3) represents an enzyme that plays an important role in the destruction of extracellular matrix macromolecules during inflammation. Its activity is controlled at several levels, including genetic transcription, and cell exportation, and activation/inhibition on extracellular matrix. On the other hand, DNA methylation represents an epigenetic event that may control the gene expression. Thus, our interesting in this study was to investigate the DNA methylation status of CpG sites located at position - 635 and - 686 on the MMP3 gene promoter from healthy gingival tissue and inflamed gingival tissue biopsied from individuals with chronic periodontitis. Method: Genomic DNA (gDNA) was purified from gingival tissue from 30 healthy individuals (control group) and 30 individuals with chronic periodontitis (periodontitis group), using the phenol protocol. The analysis of DNA methylation status was performed by Specific Methylation-Sensitive Restrictio...

Objectives: Catechol-O-methyltransferase (COMT) is an enzyme involved in the metabolic degradation of catecholamines and modification of COMT activity that modulate these neurotransmitters in pain sensitivity. The purpose of the present study was to investigate the association between COMT polymorphism (Val158Met), TMJ pain sensitivity and risk of developing Temporomandibular Joint Internal Disarrangements (TMJID). Methods: DNA was obtained from Brazilian women (334 healthy women - control group; 75 volunteers with signs of TMJID and chronic pain, and 224 with signs of TMJID but no pain - patients groups). Restriction fragment length polymorphisms of polymerase chain reaction products were used to analyze COMT polymorphism in DNA fragments. Hardy-Weinberg equilibrium was tested to determine the balance in distribution of alleles. Differences in genotype frequencies among control and patients groups were assessed by chi-square test (p<0.05). Results: The differences genotypes were...

Transgenic mouse lines in which Green Fluorescent Protein (GFP) expression is under the control of tissue- and stage-specific promoters have provided powerful experimental tools for lineage analysis. Our recent studies using pOBCol3.6GFPtpz transgenic mice showed that 3.6-GFP transgene is expressed in polarizing, functional and fully differentiated odontoblasts. These studies indicated that a pOBCol3.6GFPtpz transgenic mouse provides a unique model for studying odontoblast differentiation from progenitor population. Objectives: To examine the feasibility of using the pOBCol3.6GFPtpz mice as a model for studying pulp healing and reparative dentinogenesis. Methods: Pulp exposures were created in the first maxillary molars of the pOBCol3.6GFPtpz transgenic mice using an endodontic hand file with a 150-m-tip diameter. After exposure, pulps were capped with mineral trioxide aggregate (MTA) overlaid with light-cured composite resin. Controls included exposures that were restored with comp...