Self-assembly and mineralization of full-length human amelogenin and its functional fragments / 华西口腔医学杂志

OBJECTIVES@#To investigate the dynamic process of the self-assembly behaviors of a full-length human amelogenin (AM) and its functional fragments tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide(LRAP) @*METHODS@#The full-length human AM and its functional fragments, TRAP and LRAP, were reassembled and purified @*RESULTS@#When pH=8, the full-length human AM and TRAP assembly started spontaneously and formed "nanospheres" after 15 min.The nanospheres formed by TRAP existed independently, with a uniform size but without obvious internal structures. The full-length AM was assembled hierarchically, which formed "nanospheres" and further extended in all directions, formed a chain structure, and then aggregated into a net. The self-assembly behavior of LRAP was not obvious. Proteins mostly existed in the form of monomers without "nanosphere" formation. Only few oligomers were observed. The full-length AM was induced independently for 3 days to form rod-shaped HA crystals. TRAP and LRAP proteins were added, after 3 days the crystal elongation was obvious in the c axis, but the growth in plane A and plane B was poor.@*CONCLUSIONS@#The self-assembly and mineralization behaviors of full-length human AM, TRAP, and LRAP were consistent with the directional growth mechanism of HA crystals

Paciente masculino con cariotipo 46 XX negativo para el gen SRY y sin ambigüedad genital: reporte de un caso / Male patient 46, XX SRY -negative and unambiguous genitalia: A case report

En la mayoría de los casos, la diferenciación sexual masculina ocurre con la participación del gen SRY. Sin embargo, se pueden presentar otros genotipos excepcionales, como en el caso que se presenta en este reporte. Se trata de un paciente adulto de sexo masculino atendido en el Servicio de Paternidades del Instituto de Genética de la Universidad Nacional de Colombia. Se le hicieron los análisis del gen de la amelogenina y de repeticiones cortas en tándem (Short Tandem Repeat, STR) específicas para el gen SRY con estuches comerciales de identificación humana, así como los de cariotipo convencional e hibridación in situ fluorescente del SRY, y el estudio de microdeleciones del cromosoma Y mediante reacción en cadena de la polimerasa (PCR). Se le hizo la evaluación clínica y se le brindó asesoramiento genético. El paciente no presentaba ambigüedad genital, su cariotipo era 46 XX, y el perfil molecular era negativo para el gen SRY y positivo para el ZFY. Se le diagnosticó un trastorno de diferenciación sexual 46 XX testicular no sindrómico, una rara condición genética. Solo el 20 % de los pacientes con este diagnóstico son negativos para SRY y exhiben perfiles moleculares diversos. La información disponible parece indicar que el ZFY está relacionado con la diferenciación sexual masculina, aún en ausencia del gen SRY.

In most cases, male sexual differentiation occurs with SRY gene mediation. However, exceptional genotypes have been identified, as shown in this paper. This was a male adult patient seen at the Servicio de Paternidades, Instituto de Genética, Universidad Nacional de Colombia. The following procedures were carried out: Amelogenin gene and short tandem repeat analyses using human identification commercial kits, conventional karyotype, SRY fluorescent in situ hybridization, PCR analysis for Y chromosome microdeletions, clinical evaluation, and genetic counseling. We present an adult male with unambiguous genitalia, karyotype 46,XX, and an SRY negative and ZFY positive molecular profile. The diagnosis of nonsyndromic 46,XX testicular disorder of sex development (DSD) -a rare genetic condition- was established. Only 20 % of similarly diagnosed patients are SRY negative and exhibit diverse molecular profiles. Until now, available evidence seems to indicate that, even in the absence of SRY, the ZFY factor is involved in male sexual differentiation.

Immunohistochemical study of amelogenin binding proteins in an amelogenin point mutation mouse / Estudio inmunohistoquímico de proteínas de unión de la amelogenina en un ratón con mutación puntual de amelogenina

Amelogenin is one of the enamel matrices secreted by ameloblasts. A mutation of the amelogenin gene can cause hereditary dental enamel defects known as amelogenesis imperfecta (AI). Since lysosome-associated membrane protein-1 (LAMP-1), -3 (LAMP-3), and 78kDa glucose-related protein (Grp78) were identified as binding proteins of amelogenin, several studies have suggested the involvement of these binding proteins with the cell kinetics of ameloblasts in normal or abnormal conditions. The purpose of this study is to investigate the distribution of these amelogenin binding proteins in the ameloblast cell differentiation of mice with a point mutation of the amelogenin gene (Amelx*). The incisors of Amelx* mice had a white opaque color and the tooth surface was observed to be rough under a scanning electron microscope. Among the sequential ameloblast cell differentiation in the Amelx* mice, the shape of ameloblasts at the transition stage was irregular in comparison to those in wild-type (WT) mice. Immunostaining of Grp78 revealed that the whole cytoplasm of the transition stage ameloblasts was immunopositive for Grp78 antibody, while only the distal part of cell was positive in the WT mice. Furthermore, in the Amelx* mice, the cytoplasm of the transition stage ameloblasts was immunopositive for LAMP-1 and LAMP-3. These results suggest that Amelx* may cause the abnormal distribution of amelogenin binding proteins in the cytoplasm of ameloblasts.

La amelogenina es una de las matrices de esmalte secretadas por los ameloblastos. Una mutación del gen de amelogenina puede causar defectos hereditarios del esmalte dental conocidos como amelogénesis imperfecta (AI). Dado que la proteína de membrana asociada a lisosoma-1 (LAMP-1), -3 (LAMP-3) y la proteína relacionada con la glucosa de 78 kDa (Grp78) se identificaron como proteína de unión a amelogenina, varios estudios han sugerido la participación de estas proteínas con la cinética celular de los ameloblastos en condiciones normales o anormales. El objetivo del estudio fue investigar la distribución de LAMP-1, LAM-3 y Grp78 durante la diferenciación celular de ameloblastos de ratones con una mutación puntual del gen de amelogenina (Amelx*). Los incisivos de los ratones Amelx* presentaron un color blanco opaco y se observó en microscopio electrónico de barrido que la superficie del diente era áspera. La diferenciación celular secuencial y la forma de los ameloblastos en la etapa de transición en los ratones Amelx* fue irregular en comparación con los ratones silvestres (RS). La inmunotinción de Grp78 reveló que todo el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para el anticuerpo Grp78, mientras que solo la parte distal de la célula fue positiva en los ratones RS. Además, en ratones Amelx*, el citoplasma de los ameloblastos en etapa de transición fue inmunopositivo para LAMP-1 y LAMP-3. Estos resultados sugieren que Amelx* puede causar distribución anormal de proteínas de unión a amelogenina en el citoplasma de los ameloblastos.

Alteración del gen AMELX en amelogénesis imperfecta. Una breve revisión / Alteration of the AMELX gene in amelogenesis imperfecta. A brief review

Resumen La amelogénesis imperfecta es un grupo de trastornos de desarrollo del esmalte dental asociados principalmente con mutaciones en el gen AMELX. Clínicamente presenta diferentes fenotipos que afectan la estructura y función del esmalte, tanto de la dentición primaria como secundaria. El objetivo de este estudio fue realizar una revisión bibliográfica de las funciones y mutaciones de AMELX relacionadas con amelogénesis imperfecta. Se llevó a cabo una revisión bibliográfica en dos bases de datos: PubMed y Web of Science, usando las palabras clave “AMELX”, “amelogenina”, “amelogénesis imperfecta” y “mutación de AMELX”. Fueron revisados 40 artículos y se encontró que AMELX es el gen predominante en el desarrollo del esmalte dental y de la amelogénesis imperfecta, alterando la estructura de la amelogenina. En los últimos años se han descrito las características en el proceso de amelogénesis imperfecta con diferentes fenotipos de esmalte hipoplásico o hipomineralizado y se han reportado diferentes mutaciones, con lo que se ha determinado la secuenciación del gen y las posiciones de las mutaciones.

Abstract Amelogenesis imperfecta is a group of developmental disorders of the dental enamel that is mainly associated with mutations in the AMELX gene. Clinically, it presents different phenotypes that affect the structure and function of dental enamel both in primary and secondary dentition. The purpose of this study was to conduct a literature review on the AMELX functions and mutations that are related to amelogenesis imperfecta. A literature search was carried out in two databases: PubMed and Web of Science, using the keywords “AMELX”, “amelogenin”, “amelogenesis imperfecta” and “AMELX mutation”. Forty articles were reviewed, with AMELX being found to be the predominant gene in the development of dental enamel and amelogenesis imperfecta by altering the structure of amelogenin. In the past few years, the characteristics of the amelogenesis imperfecta process have been described with different phenotypes of hypoplastic or hypo-mineralized enamel, and different mutations have been reported, by means of which the gene sequencing and the position of mutations have been determined.

Enamel biomimetics-fiction or future of dentistry / 国际口腔科学杂志·英文版

Tooth enamel is a complex mineralized tissue consisting of long and parallel apatite crystals configured into decussating enamel rods. In recent years, multiple approaches have been introduced to generate or regenerate this highly attractive biomaterial characterized by great mechanical strength paired with relative resilience and tissue compatibility. In the present review, we discuss five pathways toward enamel tissue engineering, (i) enamel synthesis using physico-chemical means, (ii) protein matrix-guided enamel crystal growth, (iii) enamel surface remineralization, (iv) cell-based enamel engineering, and (v) biological enamel regeneration based on de novo induction of tooth morphogenesis. So far, physical synthesis approaches using extreme environmental conditions such as pH, heat and pressure have resulted in the formation of enamel-like crystal assemblies. Biochemical methods relying on enamel proteins as templating matrices have aided the growth of elongated calcium phosphate crystals. To illustrate the validity of this biochemical approach we have successfully grown enamel-like apatite crystals organized into decussating enamel rods using an organic enamel protein matrix. Other studies reviewed here have employed amelogenin-derived peptides or self-assembling dendrimers to re-mineralize mineral-depleted white lesions on tooth surfaces. So far, cell-based enamel tissue engineering has been hampered by the limitations of presently existing ameloblast cell lines. Going forward, these limitations may be overcome by new cell culture technologies. Finally, whole-tooth regeneration through reactivation of the signaling pathways triggered during natural enamel development represents a biological avenue toward faithful enamel regeneration. In the present review we have summarized the state of the art in enamel tissue engineering and provided novel insights into future opportunities to regenerate this arguably most fascinating of all dental tissues.

Regeneration of Neural Networks in Immature Teeth with Non-Vital Pulp Following a Novel Regenerative Procedure

BACKGROUND AND OBJECTIVES: Recombinant amelogenin protein (RAP) was reported to induce soft-tissue regeneration in canine infected endodontically treated permanent teeth with open apices. To characterize identities of the cells found in the RAP regenerated tissues compared to authentic pulp by identifying: 1) stem cells by their expression of Sox2; 2) nerve fibers by distribution of the axonal marker peripherin; 3) axons by their expression of calcitonin gene–related peptide (CGRP); 4) the presence of astrocytes expressing glial fibrillary acidic proteins (GFAP).METHODS: A total of 240 open-apex root canals in dogs were used. After establishment of oral contamination to the pulp, the canals were cleaned, irrigated, and 120 canals filled with RAP, and the other 120 with calcium hydroxide.RESULTS: After 1, 3, and 6 months, teeth were recovered for immune-detection of protein markers associated with native pulp tissues. Regenerated pulp and apical papilla of RAP group revealed an abundance of stem cells showing intense immunoreactivity to Sox2 antibody, immunoreactivity of peripherin mainly in the A-fibers of the odontoblast layer and immunoreactivity to CGRP fibers in the central pulp region indicative of C-fibres. GFAP immunoreactivity was observed near the odontoblastic, cell-rich regions and throughout the regenerated pulp.CONCLUSIONS: RAP induces pulp regeneration following regenerative endodontic procedures with cells identity by gene expression demonstrating a distribution pattern similar to the authentic pulp innervation. A- and C-fibers, as well as GFAP specific to astrocytic differentiation, are recognized. The origin of the regenerated neural networks may be derived from the Sox2 identified stem cells within the apical papilla.

Expression and Localization of Keap1 During Amelogenesis in the Developing Molar Germ of Rats

The objective of this study was to examine the expression pattern of Kelch-like ECH-associated protein 1 (Keap1) in the maxillary 2(nd) molar germs of rats. We used the maxillary 2(nd) molar germs in rats' pup at postnatal day 3 (bell stage), 6 (crown formation stage) and 9 (root formation stage). The investigation on mRNA and protein levels were done using reverse transcription - polymerase chain reaction and western blot. Localization of Keap 1 in the maxillary 2(nd) molar germs were revealed through immunofluorescence staining. Keap1 from the maxillary 2nd molar germs were mostly manifested on postnatal day 3 and dramatically decreased on postnatal day 6 and 9 at mRNA and protein levels, while amelogenin and ameloblastin increased during the development of maxillary 2nd molar germs. During immunofluorescence analysis, the strong immunoreactivity against Keap1 was detected in the apical side of ameloblasts at the presecretory and secretory stages. However, Keap1 expression was hardly observed in the ameloblasts at the maturation stage. These results shows that Keap1 is strongly expressed in the presecretory and secretory ameloblasts of amelogenesis, and suggest that Keap1 may be a crucial molecule for the regulatory mechanisms tasked with the formation of enamel layer.

Recombinant amelogenin regulates the bioactivity of mouse cementoblasts in vitro / 国际口腔科学杂志·英文版

Amelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng · mL) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng · mL increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng · mL) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng · mL remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng · mL rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.

Application of miniSTR Loci and Its Detection System for Degraded Materials in Forensic Medicine / 法医学杂志

OBJECTIVES@#To establish multiplex system of 16 miniSTR loci, and explore its application value for the degraded materials in forensic medicine.@*METHODS@#The multiplex system of 16 miniSTR loci was established using a six-dye fluorescence labeling technology and its application value in forensic medicine was assessed.@*RESULTS@#A six-dye fluorescence labeling miniSTR amplification kit was developed, which enabled 15 autosomal STR loci, Amelogenin locus and DYS391 to be typed simultaneously. This method showed good specificity and could provide stable and accurate typing results with a sensitivity of 50 pg. This system also provided a good test result for the normal biological sample of actual cases.@*CONCLUSIONS@#The multiplex system of 16 miniSTR loci has application value for degraded and trace materials with the advantages of high sensitivity and database compatibility, which can be used for forensic casework.

Detection and Analysis of 12 Suspected Amelogenin Allelic Loss Cases / 法医学杂志

OBJECTIVES@#To observe and analyse the Amelogenin allelic loss in parent-child identification cases, and to explore the type and mechanism of Amelogenin allelic loss as well as its influence on gender identification and solutions.@*METHODS@#After the detection by SiFaSTR™ 23plex DNA identification system, samples had the characteristics of the peak area of Amelogenin X was the same as the one of adjacent heterozygote or lower than one half of adjacent homozygote in females while Amelogenin X loss was observed in males were selected. X chromosome STR （X-STR） typing and Amelogenin X sequencing were performed. The samples with Amelogenin Y loss in males were confirmed by the detection of Y chromosome STR typing and sex-determining region of Y （SRY）. The type and rate of Amelogenin allelic loss were confirmed and calculated, and the mechanism and influence of this variation were also analysed.@*RESULTS@#Amelogenin X allelic loss was observed in one male sample, the mutation in primer-binding region was confirmed by sequencing. The suspected Amelogenin X allelic loss was observed in four female samples, but the mutation in primer-binding region was confirmed by sequencing in only one sample. Amelogenin Y allelic loss was observed in seven male samples, SRY positive cases was detected in five of them, and two were SRY negative. Y-STR type was detected in four cases of the five SRY positive cases, which was not detected in the two SRY negative cases. The rate of Amelogenin allelic loss was about 0.029%.@*CONCLUSIONS@#Amelogenin X allelic loss does not affect the gender identification, but Amelogenin Y allelic loss may cause wrong gender identification. Thus, Y-STR or SRY should be detected for gender confirmation. When Y-STR genotypes are not detected in a "male" whose SRY detection is also negative, then the chromosome karyotype analysis and sex differentiation related genes test should be taken to further confirm the gender.

Diversidad del ADN mitocondrial en restos óseos prehispánicos / Mitochondrial DNA diversity in prehispanic bone remains on the eastern Colombian Andes

Resumen Introducción. El ADN antiguo que se extrae de los restos óseos humanos permite analizar la composición genética de las poblaciones precolombinas y determinar las dinámicas poblacionales que dieron origen a la diversidad de las poblaciones contemporáneas. Objetivo. Determinar la diversidad genética y la relación con otras comunidades contemporáneas y antiguas de América, de los restos óseos asociados al Templo del Sol en Sogamoso, Colombia. Materiales y métodos. Se analizaron 13 individuos pertenecientes al periodo precolombino muisca (siglos IX-XVI d. C.), provenientes de los alrededores del Templo del Sol en Sogamoso, Boyacá, Andes orientales colombianos. Se amplificó el ADN mitocondrial (ADNmt) y se determinaron los polimorfismos de la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP) para los cuatro haplogrupos amerindios (A, B, C y D). Además, se amplificaron y analizaron los marcadores autosómicos, incluida la amelogenina, y los marcadores de los polimorfismos de repeticiones cortas en tándem (Short Tandem Repeat, STR) del cromosoma Y. Resultados. El haplogrupo A fue el linaje mitocondrial más frecuente en esta población, seguido de los haplogrupos B y C; no se detectó el haplogrupo D. Los análisis de variación genética indicaron una diversidad semejante a la de las poblaciones pertenecientes a la familia lingüística chibcha, contemporánea en Colombia y Centroamérica. Se logró hacer la determinación molecular del sexo de los individuos estudiados y compararla con los datos osteológicos. Con una sola excepción, los datos bioantropológicos y moleculares concordaron. Conclusiones. Estos resultados aportan nuevos elementos a la hipótesis del origen centroamericano de los grupos chibchas del altiplano cundiboyacense con base en marcadores genéticos, y permitieron establecer el sexo y las relaciones de parentesco.

Abstract Introduction: DNA extracted from ancient human bones allows to analyze the genetic makeup of preColumbian populations and to determine the dynamics that gave rise to the diversity of contemporary populations. Objective: To determine the genetic diversity of skeletal remains associated with the Templo del Sol (Sun Temple) and their relationship with other contemporary and ancient communities of America. Materials and methods: We analyzed 13 individuals belonging to the pre-Columbian Muisca Period (IX-XVI centuries AD) from the vicinities of the Templo del Sol (Sun Temple) (Sogamoso, Boyacá) in the eastern Colombian Andes. Mitochondrial DNA was amplified and RFLPs were performed in order to type the four traditional Amerindian haplogroups (A, B, C and D). In addition, autosomal markers including amelogenin and Y-chromosome STRs were amplified. Results: Among the observed mitochondrial lineages, haplogroup A was the most frequent, followed by haplogroups B and C; no evidence of haplogroup D was found. The genetic variation analysis indicated a similar diversity of pre-Columbian Muiscas to that of contemporary populations belonging to the Chibcha linguistic family from Colombia and Central America. Molecular sexing was accomplished and it was compared to osteological data. With only one exception, anthropological and molecular data were consistent. Conclusions: Our results contribute new genetic elements supporting the hypothesis of Central American origin of the Chibcha groups of the Cundiboyacense plateau, and allowed sex typing and kinship evaluations.

Expression of enamel proteins in rough endoplasmic reticulum and golgi complex in human dental germs / Expresión de proteínas del esmalte en retículo endoplásmico rugoso y complexo golgiensis en gérmenes dentales humanos

Tooth enamel is the hardest tissue in the body. The organic matrix configuration is provided by the main proteins amelogenin, ameloblastin and enamelysin (MMP20), an enzyme that helps to shape the matrix. The aim of this study was to determine by histochemistry the expression of amelogenin and enamelysin through the rough endoplasmic reticulum in the late stages of amelogenesis, and its expression in the Complexus golgiensis (Golgi complex / Golgi apparatus) in the early stages in human fetuses. In early stages a colocalization of both proteins inside the Golgi apparatus was found, being more evident the relationship between Golgi and amelogenin (99.92 %). In the late stage, a colocalization of both proteins and rugged endoplasmic reticulum was found. With enamelysin being more evident in relation with rough endoplasmic reticulum (99.95 %). Our findings demonstrated the presence of amelogenin and enamelysin in odontoblast and ameloblast. However, the presence of these two proteins in odontoblast remains unknown.

El esmalte dental es el tejido más duro del cuerpo. La configuración de la matriz orgánica es proporcionada por las proteínas principales amelogenina, ameloblastina y enamelisina (MMP20), una enzima que ayuda a dar forma a la matriz. El objetivo de este estudio fue determinar mediante histoquímica la expresión de amelogenina y enamelisina a través del retículo endoplasmático rugoso en las últimas etapas de la amelogénesis , y su expresión en el Complexo golgiensis en las primeras etapas de formación en fetos humanos. En las primeras etapas se observó colocalización de ambas proteínas en el interior del Complexo golgiensis, siendo más evidente la relación entre Golgi y amelogenina (99,92 %). En la última etapa, se identificó una colocalización de ambas proteínas y retículo endoplásmico rugoso. Resulto más evidente la enamelisina en relación con el retículo endoplasmático rugoso (99,95 %). Nuestros resultados demostraron la presencia de amelogenina y enamelisina en odontoblastos y ameloblastos, sin embargo se desconoce la presencia de estas dos proteínas en odontoblastos.

Study of human leucine-rich amelogenin peptide and its regulation of mineralization by cryogenic transmission electron microscopy / 华西口腔医学杂志

<p><b>OBJECTIVE</b>Recombinant human leucine-rich amelogenin peptide (LRAP) was studied by cryogenic transmission electron microscopy (TEM); evaluation focused on its self-assembly and crystal growth in vitro.</p><p><b>METHODS</b>Human LRAP was recombined through prokaryotic expression vector pCold-SUMO and transformed into Escherichia coli BL21plys to acquire purified proteins. Cryogen TEM recorded assembly and self-assembling of LRAP from pH 3.5 to pH 8.0, and the hydroxyapatite crystal growth in the mixture of LRAP protein solution and artificial saliva was observed using TEM and selected area electron diffraction.</p><p><b>RESULTS</b>More than 90% purity LRAP was expressed, purified and identified as described in methods. LRAP linked into oligomers, nanospheres, nanochains, and microribbons, whereas pH value increased from 3.5 to 8.0. Mature hydroxyapatite crystal growth was guided in artificial saliva filled with calcium phosphate.</p><p><b>CONCLUSIONS</b>LRAP is simplified amelogenin functional domain and conserved the basic characters of amelogenin such as self-assembling and inducing crystallization along c axis. In the area of acellular synthesis of hydroxyapatite using extracellular enamel matrix protein, LRAP is one of candidate repair materials for irregular hard tissue defection.
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Regeneración tisular guiada en defecto óseo de 3 paredes: enfermedad periodontal y oclusión / Guided tissue regeneration and in a three-walled osseous defect: periodontal disease and occlusion

Relacionar la importancia del éxito en regeneración tisular guiada y el correcto diagnóstico del problema, en este caso enfermedad periodontaly un contacto prematuro en ORC producto de una obturación de amalgama incorrecta. Caso clínico: tratamiento de un defecto infraóseo de3 paredes mediante la utilización de hueso de origen bovino particulado junto con proteínas derivadas de la matriz del esmalte. Tanto los parámetros clínicoscomo los radiográficos fueron evaluados al inicio, en el postquirúrgico inmediato y a los 12 meses. Conclusión: se observó un alto grado de regeneración pasados los 12 meses del tratamiento. Parecería no ser siempre necesaria la utilización de membrana colágena. Las proteínas derivadas de la matrizdel esmalte serían un sustituto de la membrana en algunos casos. Resulta fundamental el chequeo de la situación oclusal en piezas periodontalmente comprometidas...

Differential Expression of Amelogenin, Enamelin and Ameloblastin in Rat Tooth Germ Development

Tooth development shows dynamic morphological changes from the stages of cap to hard tissue formation and is strictly regulated during development. In the present study, we compared expression and localization of 3 major enamel matrix proteins in rats: amelogenin, enamel and ameloblastin. DD-PCR and RT-PCR revealed differential expression of the major proteins from the cap stage to root stage. Immunofluorescence staining results indicated that amelogenin was not detected in either inner enamel epithelium or reduced enamel epithelium, but highly immunoreactive in preameloblasts and ameloblasts; in addition, it was sporadically expressed in preodontoblasts abutting preameloblasts. Ameloblastin expression was also observed in not only differentiated ameloblasts but also osteoblasts. Immunoreactivity to ameloblastin in ameloblasts was strong in Tomes' processes. Enamelin was exclusively localized along the entire newly formed and maturing enamel. Enamelin was largely localized in near Tomes' processes and enamel rods in maturing enamel. Alendronate treatment resulted in down-regulation of amelogenin and ameloblastin at both transcription and translation levels; whereas, enamelin expression was unchanged in response to the treatment. These results suggested that amelogenin, ameloblastin and enamelin might be implicated in cell differentiation, adhesion of ameloblasts to enamel and enamel crystallization during enamel matrix formation, respectively.

Application of the Peak Area Ratio of STR Loci to Amelogenin Locus in the Estimation of DNA Degradation / 法医学杂志

OBJECTIVE@#To explore the change rules of peak area ratio of STR loci to Amelogenin (AMEL) locus (STR/AMEL), a sex-determining gene in DNA degradation, and to evaluate the application of STR/AMEL value in the estimation of DNA degradation degree.@*METHODS@#DNA was extracted from iliopsoas, and the variations of STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) were analyzed after the artificial degradation was made by DNase I, and the changes of these three ratios of the iliopsoas naturally degraded in an outdoor environment were also analyzed. The regression curves were analyzed using the periods of DNA degradation and outside the body as the independent variable (x) and the STR/AMEL value as the dependent variable (y) and three curve equations under two conditions were established.@*RESULTS@#Both under the conditions of artificial and natural degradation, STR/AMEL value had a negative relationship with the degradation time. The relationship between STR/AMEL and degradation time can be well simulated by the cubic function. R2 was over 0.99 under controlled degradation condition and over 0.86 under natural degradation condition.@*CONCLUSION@#The STR/AMEL value (Penta E/AMEL, Penta D/AMEL, FGA/AMEL) is negatively related with the DNA degradation degree, which follows mathematical regression models strictly, and it might be applied to evaluate the DNA degradation degree.

Research Progress on Gene Alterations of Amelogenin Locus in Gender Identification / 法医学杂志

There are two kinds of amelogenin gene mutation, including mutation in primer-binding region of amelogenin gene and micro deletion of Y chromosome encompassing amelogenin gene, and the latter is more common. The mechanisms of mutation in primer-binding region of amelogenin gene is nucleotide point mutation and the mechanism of micro deletion of Y chromosome encompassing amelogenin gene maybe non-allelic homologous recombination or non-homologous end-joining. Among the population worldwide, there is a notably higher frequency of amelogenin gene mutations in Indian population, Sri Lanka population and Nepalese population which reside within the Indian subcontinent. Though amelogenin gene mutations have little impact on fertility and phenotype, they might cause incorrect result in gender identification. Using composite-amplification kit which including autosomal STR locus, amelogenin gene locus and multiple Y-STR locus, could avoid wrong gender identification caused by amelogenin gene mutation.

Recognition of Y Fragment Deletion by Genotyping Graphs after Amplified by PowerPlex® 21 Detection Kit / 法医学杂志

OBJECTIVES@#To recognize the possibility of Y fragment deletion of Amelogenin gene intuitively and simply according to the genotyping graphs.@*METHODS@#By calculating the ratio of total peak height of genotyping graphs, the statistics of equilibrium distribution between Amelogenin and D3S1358 loci, Amelogenin X-gene and Amelogenin Y-gene, and different alleles of D3S1358 loci from 1 968 individuals was analyzed after amplified by PowerPlex® 21 detection kit.@*RESULTS@#Sum of peak height of Amelogenin X allele was not less than 60% that of D3S1358 loci alleles in 90.8% female samples, and sum of peak height of Amelogenin X allele was not higher than 70% that of D3S1358 loci alleles in 94.9% male samples.@*CONCLUSIONS@#The result of genotyping after amplified by PowerPlex® 21 detection kit shows that the possibility of Y fragment deletion should be considered when only Amelogenin X-gene of Amelogenin is detected and the peak height of Amelogenin X-gene is not higher than 70% of the total peak height of D3S1358 loci.

Viabilidade do derivado da matriz do esmate no tratamento de recessões classes I e II de Miller / Viability of the enamel matrix derivate in the treatment of the recessions class I and II of Miller

A recessão gengival é uma alteração comum que gera transtornos funcionais e estéticos nos indivíduos acometidos. Na busca por uma melhor resolução dessa condição, diversos métodos de tratamento têm sido desenvolvidos ao longo das últimas três décadas, sendo que, atualmente, as técnicas e os biomateriais regenerativos estão em evidência. O presente artigo é uma revisão da literatura científica, pertinente sobre o potencial clínico da utilização do derivado da matriz do esmalte, composto 90% por amelogeninas, no tratamento das recessões gengivais classes I e II de Miller.

Triagem de mutações e polimorfismos de genes candidatos à malformação dentária em pacientes com fissura labiopalatinas / Screening of mutations and polymorphisms of candidate genes to dental malformation in patients with cleft lip and palate

O propósito deste trabalho foi investigar a ocorrência de mutações e polimorfismos em genes candidatos aos defeitos na formação do esmalte dentário em indivíduos com fissura labiopalatina (FLP) transforame incisivo unilateral ou bilateral isolada e associar o genótipo-fenótipo dos indivíduos com FLP e malformação dentária (MD) nos dentes incisivos centrais superiores permanentes. Foram coletadas amostras de saliva de 165 indivíduos de 6 a 15 anos de idade, de ambos os sexos, divididos em 4 grupos de estudo: Grupo 1 - 46 indivíduos com FLP e MD; Grupo 2 - 34 indivíduos com FLP e sem MD; Grupo 3 - 34 indivíduos sem FLP e com MD; Grupo 4 - 51 indivíduos sem FLP e MD. Foi realizada a extração do DNA genômico das amostras de saliva, seguida da Reação em Cadeia da Polimerase, sequenciamento direto dos éxons 2, 3, 4, 5, 6 e 7 do gene AMELX e genotipagem dos SNPs rs3796703, rs3796704, rs3796705, rs7671281, rs2609428 e rs35951442 no gene ENAM. Para a análise estatística dos resultados foi utilizado o Teste Exato de Fisher e o Teste do Qui-quadrado de Pearson. Em relação ao sequenciamento direto do gene AMELX, mutações foram encontradas em 30,4% (n=14), 35,3% (n=12), 11,8% (n=4) e 13,7% (n=7) dos indivíduos dos Grupos 1, 2, 3 e 4, respectivamente. Trinta e sete mutações foram detectadas e distribuídas ao longo dos éxons 2 (1 mutação - 2,7%), 6 (30 mutações - 81,08%) e 7 (6 mutações - 16,22%) do gene AMELX. Houve um aumento significativo (p=0,003) na frequência de mutações nos indivíduos com FLP (Grupos 1 e 2 - 65,7%) em relação aos indivíduos sem FLP (Grupos 3 e 4 - 25,5%). Em relação às 30 mutações encontradas no éxon 6, 43,34% (n=13), 23,33% (n=7), 13,33% (n=4) e 20% (n=6) foram encontrados nos Grupos 1, 2, 3 e 4, respectivamente. A mutação silenciosa c.261C>T (rs2106416) foi detectada em 26 indivíduos distribuídos nos quatro grupos estudados, sendo significativamente mais encontrada (p=0,003) nos grupos com FLP (23,75%), em comparação com os...

The purpose of this study was to investigate the occurrence of mutations and polymorphisms (SNPs) in candidate genes to defects in the formation of enamel in individuals with cleft lip and palate (CLP) unilateral or bilateral incisive transforame isolated and associate genotype-phenotype of individuals with CLP and dental malformation (DM) in permanent teeth maxillary central incisors. For analysis of the proposed genes, saliva samples from 165 individuals from 6 to 15 years old, of both genders, were collected and divided into 4 groups: Group 1 - 46 individuals with CLP and DM; Group 2 - 34 individuals with CLP and without DM; Group 3 - 34 subjects without CLP and DM; Group 4 - 51 subjects without CLP and DM. Extraction of genomic DNA from saliva samples was performed, followed by Polymerase Chain Reaction, direct sequencing of 2, 3, 4, 5, 6 and 7 exons of AMELX gene and genotyping of SNPs rs3796703, rs3796704, rs3796705, rs7671281, rs2609428 and rs35951442 in the ENAM gene. For statistical analysis we used the Fisher's exact test and Pearson's chi-square test. Regarding direct sequencing of AMELX gene, mutations were found in 30.4% (n=14), 35.3% (n=12), 11.8% (n=4) and 13.7% (n=7) of individuals in Groups 1, 2, 3 and 4, respectively. Thirty-seven mutations were detected and distributed over the exons 2 (1 mutation - 2.7%), 6 (30 mutations - 81.08%) and 7 (6 mutations - 16.22%) of AMELX gene. There was a significant increase (p=0.003) in the frequency of mutations in individuals with CLP (Groups 1 and 2 - 65.7%) compared to subjects without CLP (Groups 3 and 4 - 25.5%). Regarding the 30 mutations found in exon 6, 43.34% (n=13), 23.33% (n=7), 13.33% (n=4) and 20% (n=6) were found in Groups 1, 2, 3 and 4, respectively. The c.261C>T silent mutation (rs2106416) was detected in 26 individuals distributed in all groups studied, and was significantly more found (p=0.003) in the groups with CLP (23.75%) compared to the groups without CLP (8.23%). In groups without...