Estimating the Influence of Physicochemical and Biochemical Property Indexes on Selection for Amino Acids Usage in Eukaryotic Cells.

Proteins can evolve by accumulating changes on amino acid sequences. These changes are mainly caused by missense mutations on its DNA coding sequences. Mutations with neutral or positive effects on fitness can be maintained while deleterious mutations tend to be eliminated by natural selection. Amino acid changes are influenced by the biophysical, chemical, and biological properties of amino acids. There is a multiplicity of amino acid properties that can influence the function and expression of proteins. Amino acid properties can be expressed into numerical indexes, which can help to predict functional and structural aspects of proteins and allow statistical inferences of selection pressure on amino acid usage. The accuracy of these analyses may be compromised by the existence of several numerical indexes that measure the same amino acid property, and the lack of objective parameters to determine the most accurate and biologically relevant index. In the present study, the gradient consistency test was used in order to estimate the magnitude of directional selection imparted by amino acid biochemical and biophysical properties on protein evolution.

Effects of Pamidronate on Dental Enamel Formation Assessed by Light Microscopy, Energy-Dispersive X-Ray Analysis, Scanning Electron Microscopy, and Microhardness Testing.

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.

Translational signatures and mRNA levels are highly correlated in human stably expressed genes.

BACKGROUND: Gene expression is one of the most relevant biological processes of living cells. Due to the relative small population sizes, it is predicted that human gene sequences are not strongly influenced by selection towards expression efficiency. One of the major problems in estimating to what extent gene characteristics can be selected to maximize expression efficiency is the wide variation that exists in RNA and protein levels among physiological states and different tissues. Analyses of datasets of stably expressed genes (i.e. with consistent expression between physiological states and tissues) would provide more accurate and reliable measurements of associations between variations of a specific gene characteristic and expression, and how distinct gene features work to optimize gene expression. RESULTS: Using a dataset of human genes with consistent expression between physiological states we selected gene sequence signatures related to translation that can predict about 42% of mRNA variation. The prediction can be increased to 51% when selecting genes that are stably expressed in more than 1 tissue. These genes are enriched for translation and ribosome biosynthesis processes and have higher translation efficiency scores, smaller coding sequences and 3' UTR sizes and lower folding energies when compared to other datasets. Additionally, the amino acid frequencies weighted by expression showed higher correlations with isoacceptor tRNA gene copy number, and smaller absolute correlation values with biosynthetic costs. CONCLUSION: Our results indicate that human gene sequence characteristics related to transcription and translation processes can co-evolve in an integrated manner in order to optimize gene expression.

Association of matrix metalloproteinase gene polymorphism with temporomandibular joint degeneration.

Temporomandibular joint (TMJ) degeneration is a frequent cause of orofacial pain. Matrix metalloproteinases (MMPs) degrade extracellular matrix components and play an important role in TMJ degeneration. We investigated the frequency of the MMP1 1G/2G polymorphism (rs1799750), the MMP3 5A/6A polymorphism (rs3025058), and the MMP9 C/T polymorphism (rs3918242) in individuals with TMJ degeneration, in order to analyze the association of polymorphisms in these genes with TMJ degeneration. The population studied comprised 117 healthy controls and 115 individuals diagnosed with TMJ degeneration upon examination of magnetic resonance imaging (MRI) and computed tomography (CT) images. Genotypes were determined using PCR restriction fragment length polymorphism (RFLP). Logistic regression analyses revealed an association between the MMP1 2G/2G genotype and degeneration; in contrast, there was no association between either the MMP3 or the MMP9 genotype and degeneration. Our results may indicate a role for the MMP1 polymorphism in TMJ degeneration.

Birefringence of the secretory-stage enamel organic extracellular matrix from rats submitted to successive injections of bisphosphonates.

The aim of the present study was to assess birefringence of the secretory-stage enamel organic extracellular matrix (ECM) and mechanical properties of mature enamel from rats treated with bisphosphonates. Longitudinal sections were obtained from upper incisors of rats that had been submitted to injections of bisodic etidronate (8 mg/Kg/day), sodium alendronate (30 microg/Kg/day), or sodium chloride as control (8 mg/Kg/day) for 42 days. Sections were immersed in 80% glycerin for 30 min and optical retardation of birefringence brightness in the secretory-stage enamel organic ECM was determined in nanometers. Etidronate-treated rats exhibited extensive morphological changes in the secretory-stage enamel organic ECM inclusive nonbirefringent conspicuous incremental lines, but presented optical retardation values similar to those showed by control rats (p > 0.05). Birefringence of secretory enamel organic ECM from etidronate rats presented an irregular aspect. Alendronate-treated rats did not show morphological alterations in the secretory-stage enamel organic ECM, however, they presented significant reduction in optical retardation of birefringence brightness when compared with control and etidronate rats (p < 0.01). Alendronate and etidronate groups exhibited reductions of approximately 6-10% in mature enamel cross-sectional microhardness when compared with control group (p < 0.01). Scanning electron microscopy analysis showed extensive alterations in mature enamel only from etidronate group with absence of enamel rods. The present work shows that bisphosphonates can affect the birefringence of the secretory-stage enamel organic ECM. The results presented here suggest that alterations in the supramolecular organization of the secretory-stage enamel organic ECM are a plausible mechanism by which environmental factors may cause enamel defects.

Transcriptional activity analysis of promoter region of human PAX9 gene under dexamethasone, retinoic acid, and ergocalciferol treatment in MCF-7 and MDPC23.

PAX9 gene is a member of the family homeobox of transcription factors and performs important function in development and organogenesis. Mutations in PAX9 coding sequences have been implicated in autosomal dominant oligodontia affecting predominantly permanent molars and second premolars. Previous studies have shown that PAX9 is required for secondary palate development and teratogens have been identified as inducers of a tooth and craniofacial malformations. This work focused on the analysis on the 5'-flanking region of the PAX9 gene studying the influence of retinoic acid, dexamethasone, and vitamin D on the expression of PAX9 by expression constructs that carry the reporter gene luciferase. As results, retinoic acid and dexamethasone showed progressive decrease of PAX9 expression. PAX9-pGL3B1 and PAX9-pGL3B2 promoter was inhibited under the treatment of dexamethasone and ergocalciferol. Retinoic acid and dexamethasone did not alter PAX9-pGL3B3 behavior indicating that sequences present between -1106 and +92 were important for the transcriptional activity of PAX9 promoter. In this study, we characterized the transcriptional activity of specific regions of the PAX9 promoter gene and we demonstrated that retinoic acid and ergocalciferol can modulate the transcriptional activity of PAX9 gene.

DNA methylation status of the IL8 gene promoter in oral cells of smokers and non-smokers with chronic periodontitis.

AIM: This study analysed the status of DNA methylation in the promoter region of the IL8 gene in oral mucosa cells from healthy, smoker and non-smoker subjects with chronic periodontitis and compared these findings among groups with mRNA levels. MATERIAL AND METHODS: Genomic DNA from epithelial oral cells of 41 healthy subjects, 30 smokers with chronic periodontitis and 40 non-smokers with chronic periodontitis were purified and modified by sodium bisulphite. Genomic DNA from blood leucocytes and gingival cells from biopsies of 13 subjects of each group were also purified and modified by sodium bisulphite. Modified DNA was submitted by methylation-specific polymerase chain reaction (PCR) (MSP), electrophoresed on 10% polyacrylamide gels and stained with SYBR Gold. Total RNA from gingival cells was also isolated using the TRIzol reagent, and real-time PCR performance was used to detect the levels of interleukin-8 mRNA. RESULTS: Our results indicate that individuals with chronic periodontitis, independent of smoking habit, have a higher percentage of hipomethylation of the IL8 gene than those controls in epithelial oral cells (p<0.0001), and expression of higher levels of interleukin-8 (IL-8) mRNA than controls in gingival cells (p=0.007). No significant differences among groups were observed in gingival cells and blood cells. CONCLUSION: We conclude that inflammation in the oral mucosa might lead to changes in the DNA methylation status of the IL8 gene in epithelial oral cells.

2-hydroxyethyl methacrylate as an inhibitor of matrix metalloproteinase-2.

This study evaluated the effect of different concentrations of 2-hydroxyethyl methacrylate (HEMA) on the inhibition of matrix metalloproteinase-2 (MMP-2) in vitro. Mouse gingival explants were cultured overnight in Dulbecco's modified Eagle's minimal essential medium, following which the expression of secreted enzymes was analyzed by gelatin zymography and the effects of different amounts of HEMA on enzyme activity were investigated. The gelatinolytic proteinases present in the conditioned media were characterized as being matrix metalloproteinases (MMPs) by means of specific chemical inhibition. The MMPs present in the conditioned media were identified, using immunoprecipitation, as MMP-2. Three major bands were detected in the zymographic assays and were characterized, according to their respective molecular weights, into the following forms of MMP-2: zymogene (72 kDa), intermediate (66 kDa), and active (62 kDa). All forms of MMP-2 were inhibited by HEMA in a dose-dependent manner, implying that MMP-2 may be inhibited by HEMA in vivo.

Novel mutations in the IRF6 gene in Brazilian families with Van der Woude syndrome.

Van der Woude Syndrome (VWS) is an autosomal craniofacial disorder characterized by lower lip pits and cleft lip and/or palate. Mutations in the interferon regulatory factor 6 (IRF6) gene have been identified in patients with VWS. To identify novel IRF6 mutations in patients affected by VWS, we screened 2 Brazilian families, sequencing the entire IRF6-coding region and flanking intronic boundaries. Two novel heterozygous mutations were identified: a frame shift mutation with deletion of G at the nucleotide position 520 in the exon 6 (520delG), and a missense single nucleotide substitution from T to A at nucleotide position 1135 in exon 8 (T1135A). By using restriction enzyme analysis, we were able to demonstrate the lack of similar mutations in unrelated healthy individuals and non-syndromic cleft lip and palate patients. Our results further confirmed that haploinsufficiency of the IRF6 gene results in VWS.

Extraction of genomic DNA from paraffin-embedded tissue sections of human fetuses fixed and stored in formalin for long periods.

The advent of polymerase chain reaction (PCR) technology has increased the interest in fetal specimens housed in anatomy museums, as they may represent a unique source of genetic material for the study of uncommon or rare pathological conditions such as congenital malformations, neoplastic processes and parasitic as well as other infectious diseases. The aim of this study is to evaluate the quality of genomic DNA extracted from paraffin-embedded tissue sections of human fetuses that have been maintained in formalin for several years. Fetal tissues were embedded in paraffin, and tissue sections were submitted to ethanol/xylene dewaxing, followed by DNA extraction with ammonium acetate. DNA fragments were amplified from DNA extracted from formalin-fixed tissue sections, but not from Bouin-fixed tissues (average yield of 13.7 microg/ml from 10 umbilical cord sections of 10 microm; A(260):A(280)=1.55,). The addition of bovine serum albumim increased the yield of PCR amplification. Genomic DNA can be reliably amplified from paraffin-embedded human fetal tissues that had been fixed in formalin during 19 years and used for microdissection studies. This simple, cost-effective, and non-laborious method should facilitate the molecular analysis of a large number of specimens fixed for long periods of time.

Osseointegrated implant failure associated with MMP-1 promotor polymorphisms (-1607 and -519).

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Automated biometrics-based personal identification of the Hunter-Schreger bands of dental enamel.

The use of automated biometrics-based personal identification systems is a ubiquitous procedure in present times. Biometrics has certain limitations, such as in cases when bodies are decomposed, burned, or only small fragments of calcified tissues remain. Dental enamel is the most mineralized tissue of organisms and resists post-mortem degradation. It is characterized by layers of prisms of regularly alternating directions, known as Hunter-Schreger bands (HSB). In this article, we show that the pattern variation of the HSB, referred here as toothprint, can be used as a biometric-based parameter for personal identification in automated systems.

Association between PAX-9 promoter polymorphisms and hypodontia in humans.

Hypodontia, the congenital absence of one or a few teeth, is one of the most common alterations of the human dentition. The most common permanent missing teeth are the third molars, second premolars, and maxillary lateral incisors. Hypodontia does not represent a serious public health problem, but it may cause masticatory and speech dysfunctions, and esthetic problems. PAX 9 is believed to play an important role in tooth development. It is expressed at initiation, bud, cap, and bell stages of odontogenesis. Mutations in PAX 9 coding sequences have been implicated in autosomal dominant oligodontia affecting predominantly permanent molars and second premolars. Here, we report two polymorphisms in the promoter region of PAX 9 gene that are associated with hypodontia. DNA was extracted from buccal epithelial cells of 106 healthy Control individuals and of 102 unrelated individuals with hypodontia. PCR-RFLP was employed in the investigation of G-1031 A and T-912 C polymorphisms. Significant differences were obtained comparing Control and Test groups. Alleles G and T were found at a significant higher frequency in individuals with hypodontia, whereas alleles A and C were more frequent in Control subjects, p=0.0094 and 0.0086, respectively. The GT haplotype was significantly more prevalent in the hypodontia group, while the AC haplotype was more frequent in the Control group. These results indicate that polymorphisms in the promoter region of PAX 9 gene may have an influence on the transcriptional activity of this gene and are associated with hypodontia in humans.

HaCaT anchorage blockade leads to oxidative stress, DNA damage and DNA methylation changes.

Cell adhesion plays an important role in neoplastic transformation. Thus, anchorage-independent growth and epithelial-mesenchymal transition, which are features associated to anoikis-resistance, are vital steps in cancer progression and metastatic colonization. Cell attachment loss may induce intracellular oxidative stress, which triggers DNA damage as methylation changes. HaCaT lineage cells were submitted to periods of 1, 3, 5 and 24 h of anchorage blockage with the purpose of study of oxidative stress effect on changes in the DNA methylation pattern, derived from attachment blockade. Through this study, HaCaT anchorage blockage-induced oxidative stress was reported to mediate alterations in global DNA methylation changes and into TP53 gene promoter pattern during anoikis-resistance acquisition. Furthermore, at the first experimental time-periods (1, 3 and 5 h), genome hypermethylation was found; however, genome hypomethylation was observed in later time-periods (24 h) of attachment impediment. The TP 53 methylation analyses were performed after 24 h of replated anoikis-resistance cells and same methylation pattern was observed, occurring an early (1 and 3 h) hypermethylation that was followed by late (5 and 24 h) hypomethylation. However, LINE-1, a marker of genomic instability, was perceived in time-dependent hypomethylation. The mRNA levels of the DNMTs enzymes were influenced by cell attachment blockage, but non-conclusive results were obtained in order to match DNMTs transcription to pattern methylation results. In conclusion, DNA damage was found, leaded by oxidative stress that has come up from HaCaT anchorage blockade, which rises a global genome hypomethylation tendency as consequence, which might denote genomic instability.

Effect of lead on dental enamel formation.

In this work the effects of lead toxicity on dental enamel formation were studied. Epidemiological data and animal studies show an association between lead exposure and higher caries prevalence, but the mechanism underlying this association is still unknown. Here we present data on enamel formation in rats exposed to lead for 70 days in the drinking water. Enamel matrix was used for protein analysis and dry weight determination, while mature enamel was used for microhardness testing. Enamel matrix was scraped from the teeth and analyzed by electrophoresis in bulk or according to developing stage. Increased amounts of protein were observed in animals exposed to lead when the same weight of matrix was electrophoresed by protein electrophoresis. When extracts were prepared according to developing stages, no differences in the amount of protein or band pattern were observed. Upper incisors were cut longitudinally for Knoop enamel microhardness determination in four regions of the teeth. Microhardness analysis revealed statistically significant (P<0.05) decrease in the microhardness values of enamel from rats exposed to lead in regions of maturation but not of fully mature enamel. These results indicate a delay in enamel mineralization in incisor teeth from animals exposed to lead, highlighting a potentially important effect of lead toxicity not yet explored.

Analysis of MMP-1 and MMP-9 promoter polymorphisms in early osseointegrated implant failure.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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). DISCUSSION AND CONCLUSION: 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.

Comparison of microtensile bond strength to enamel and dentin of human, bovine, and porcine teeth.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Absence of association between transforming growth factor-beta1 promoter polymorphisms and hypodontia.

Hypodontia, the congenital absence of one or a few teeth, is one of the most common alterations of the human dentition. The most common permanent missing teeth are the third molars, second premolars, and maxillary lateral incisors. Although hypodontia does not represent a serious public health problem, it may cause masticatory and speech dysfunctions and esthetic problems. Transforming growth factor-beta1 (TGF-beta1) is believed to play an important role in tooth development. Its gene is expressed at bud, cap, and bell stages of odontogenesis. Genetic polymorphisms in the TGF-beta1 gene promoter were shown to interfere with the transcriptional activity of this gene. To further investigate the role of the TGF-beta1 gene in human hypodontia, we analyzed the frequencies of the -509 polymorphism (C-T) alleles and -800 polymorphism (G-A) alleles and genotypes in the TGF-beta1 gene promoter in 51 Caucasian subjects with hypodontia and 48 control individuals. Our data suggest that these TGF-beta1 promoter polymorphisms are not associated with hypodontia.

The role of modularity in the evolution of primate postcanine dental formula: integrating jaw space with patterns of dentition.

The assembly of a phenotype into modules or developmental fields, which are semiautonomous units in development and function, seems to be one of the strategies to increase the capacity to produce phenotypic variation. In mammals the upper dentition is formed on two distinct developmental units, wherein incisors are formed on the primary palate, which is derived from the embryonic frontonasal process, and the other teeth (canine, premolar, and molar) are formed on the alveolar bone, which is derived from the maxillary process (termed herein as PALATE2). The aim of the present work was to analyze the variations in size and number of premolar and molar teeth in primate dentition and to correlate these morphometrical parameters with the relative size of these tooth classes with respect to PALATE2. Furthermore, we seek to understand to what extent the changes in the relative size of premolar and molar fields can influence the size of each tooth within its respective field, and how these parameters connect with the variations in the dental formula that occurred during primate evolution. The data presented here not only indicate that premolar and molar fields can be seen as submodules of a larger and hierarchically superior module (i.e., PALATE2) but also present quantitative parameters that allow us to understand how variations in the relative size of premolar and molar teeth connect with the variations in the dental formula that occurred during primate evolution.

Interleukin-8 gene promoter polymorphism (rs4073) may contribute to chronic periodontitis.

BACKGROUND: The proinflammatory chemokine interleukin (IL)-8 is important in the regulation of the inflammatory response. Analyses of the single nucleotide polymorphism (SNP) reference sequence (rs) 4073 showed that the A allele upregulated IL-8 levels after stimulation with lipopolysaccharides. We investigated the association of the SNP rs4073 with chronic periodontitis. METHODS: Genotyping was performed by a standard polymerase chain reaction-restriction fragment length polymorphism assay in 289 genomic DNA samples of healthy control subjects and patients with chronic periodontitis; analyses were adjusted by multivariate logistic regression modeling. A real-time polymerase chain reaction performance was used to detect levels of the IL-8 mRNA. RESULTS: The analysis pointed to a statistically significant association of chronic periodontitis with the heterozygous TA genotype (P = 0.001); the results showed an increase in the frequency of the A allele in the diseased group (36% in the control group versus 48% in the periodontitis group). The higher levels of the IL-8 mRNA were found in the periodontitis group, mainly in individuals who presented the TA genotype (P = 0.03). CONCLUSION: The SNP rs4073 was associated with chronic periodontitis in non-smoker Brazilian subjects because the frequency of the A allele was higher in the disease group than in the control group, and the TA genotype was associated with increased levels of IL-8 mRNA transcripts.