RESUMO
The hyline tribe Lophyohylini includes 87 species of treefrogs, of which cytogenetics aspects have been studied in less than 20% of them. In order to evaluate the evolution of some of its chromosome characters (NOR position, C-bands, and DAPI/CMA3 bands), we studied the karyotypes of 21 lophyohylines, 16 of them for the first time, and analyzed them in a phylogenetic context. Most species showed similar karyotypes regarding chromosome number (2n = 24) and morphology (FN = 48), excepting Phyllodytes edelmoi and Osteocephalus buckleyi with 2n = 22 (FN = 44) and 2n = 28 (FN = 50), respectively. The NOR location was variable among species and provided valuable phylogenetic information. This marker was located in pair 11 in all species of Trachycephalus, Itapotihyla langsdorffii, and Nyctimantis arapapa, representing the plesiomorphic condition of Lophyohylini. Besides, other apomorphic states were recovered for the clades comprising N. rugiceps and N. siemersi (NOR in pair 5), and Dryaderces pearsoni, Osteocephalus, and Osteopilus (NOR in pair 9). Phyllodytes presented variation for NORs position; they were in pair 2 in P. edelmoi, pair 7 in P. melanomystax, and pair 8 in P. gyrinaethes and P. praeceptor. Polymorphisms in size, number, and activity of this marker were observed for N. siemersi, Osteocephalus fuscifacies, and some species of Trachycephalus. Remarkably, in N. siemersi NORs were detected on a single chromosome in the two specimens studied by this technique, raising the question of how this complex polymorphism is maintained. Interstitial telomeric sequences were found in P. edelmoi, P. melanomystax, and Osteocephalus buckleyi, and their presence seems to be not related to the chromosome reorganization events. Finally, some species showed spontaneous rearrangements, possibly as a consequence of an uncommon phenomenon in anuran cytogenetics: the presence of fragile sites or secondary constrictions not associated with NORs. We propose that this rare feature would have played an important role in the evolution of this group of frogs. From the evidence obtained in this and previous studies, we conclude that Lophyohylini presents a complex chromosome evolution.
Assuntos
Anuros/genética , Cromossomos/genética , Animais , Anuros/classificação , Bandeamento Cromossômico , Sítios Frágeis do Cromossomo/genética , Cromossomos/ultraestrutura , Análise Citogenética , Evolução Molecular , Feminino , Cariótipo , Masculino , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/ultraestrutura , Filogenia , Polimorfismo Genético , América do Sul , Especificidade da Espécie , Telômero/genéticaRESUMO
Congenital transmission of Trypanosoma cruzi (T. cruzi) is partially responsible for the progressive globalization of Chagas disease. During congenital transmission the parasite must cross the placental barrier where the trophoblast, a continuous renewing epithelium, is the first tissue in contact with the parasite. The trophoblast turnover implies cellular proliferation, differentiation and apoptotic cell death. The epithelial turnover is considered part of innate immunity. We previously demonstrated that T. cruzi induces cellular differentiation and apoptosis in this tissue. Here we demonstrate that T. cruzi induces cellular proliferation in a trophoblastic cell line. We analyzed the cellular proliferation in BeWo cells by determining DNA synthesis by BrdU incorporation assays, mitotic index, cell cycle analysis by flow cytometry, as well as quantification of nucleolus organizer regions by histochemistry and expression of the proliferation markers PCNA and Ki67 by Western blotting and/or immunofluorescence. Additionally, we determined the ERK1/2 MAPK pathway activation by the parasite by Western blotting.
Assuntos
Proliferação de Células , Trofoblastos/citologia , Trofoblastos/parasitologia , Trypanosoma cruzi/fisiologia , Animais , Divisão Celular , Linhagem Celular Tumoral , DNA/biossíntese , Citometria de Fluxo , Fase G2 , Antígeno Ki-67/metabolismo , Sistema de Sinalização das MAP Quinases , Índice Mitótico , Região Organizadora do Nucléolo/ultraestrutura , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fase S , Trofoblastos/metabolismoRESUMO
We conducted a cytogenetic study of four hyline frog species (Dendropsophus elegans, D. microps, D. minutus and D. werneri) from southern Brazil. All species had 2n = 30 chromosomes, with interspecific and intraspecific variation in the numbers of metacentric, submetacentric, subtelocentric and telocentric chromosomes. C-banding and fluorochrome staining revealed conservative GC-rich heterochromatin localized in the pericentromeric regions of all species. The location of the nucleolus organizer regions, as confirmed by fluorescent in situ hybridization, differed between species. Telomeric probes detected sites that were restricted to the terminal regions of all chromosomes and no interstitial or centromeric signals were observed. Our study corroborates the generic synapomorphy of 2n = 30 chromosomes for Dendropsophus and adds data that may become useful for future taxonomic revisions and a broader understanding of chromosomal evolution among hylids.
Assuntos
Anuros/genética , Centrômero/ultraestrutura , Heterocromatina/ultraestrutura , Região Organizadora do Nucléolo/ultraestrutura , Telômero/ultraestrutura , Animais , Evolução Biológica , Brasil , Bandeamento Cromossômico , Feminino , Florestas , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Ploidias , Especificidade da EspécieRESUMO
BACKGROUND: Prevention and early diagnosis have the greatest potential for public health and are the most effective method in the long-term to control oral cancer. The aim was to apply PAP staining together with AgNOR staining and morphometric analysis in oral exfoliative cytology, to determine the sensitivity and specificity of these methods in the detection of malignant changes for the purposes of both initial population monitoring and follow-up. METHODS: AgNOR, Papanicolau, and morphometric tests were conducted in samples of patients with oral cancer, oral potentially malignant disorders and controls (opposite side of lesions). Specificity and sensitivity values for each stain method and the curve under ROC area were estimated. RESULTS: The diagnostic variables which allowed greatest accuracy in identifying malignancy relative to the healthy control were cluster (76.92%), satellite (75.64%), and total (90%). The diagnosis was seen to be associated with PAP and total AgNOR, total AgNOR and PAP, total AgNOR and satellites and clusters, and total AgNOR nuclear area/cytoplasmic area ratio. CONCLUSIONS: The total number of AgNOR is a reliable marker for detecting neoplastic cells; this method increases sensitivity and specificity by decreasing the likelihood of false negatives or positives, as the accuracy obtained was 90%. It is also a low-cost, non-invasive, simple methodology that can be recommended to help the early detection of oral cancer and monitoring of patients with a first diagnosis of cancer.
Assuntos
Corantes , Citodiagnóstico/métodos , Detecção Precoce de Câncer/métodos , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos Nucleares/ultraestrutura , Área Sob a Curva , Núcleo Celular/ultraestrutura , Estudos Transversais , Citodiagnóstico/estatística & dados numéricos , Citoplasma/ultraestrutura , Detecção Precoce de Câncer/estatística & dados numéricos , Feminino , Humanos , Leucoplasia Oral/diagnóstico , Leucoplasia Oral/patologia , Líquen Plano Bucal/diagnóstico , Líquen Plano Bucal/patologia , Masculino , Pessoa de Meia-Idade , Região Organizadora do Nucléolo/ultraestrutura , Teste de Papanicolaou/estatística & dados numéricos , Curva ROC , Sensibilidade e Especificidade , Coloração pela Prata/estatística & dados numéricos , Adulto JovemRESUMO
AIM: To evaluate the proliferative potential and the cell proliferation rate of odontogenic epithelial cells. MATERIALS AND METHODS: Forty-two cases of pericoronal follicles of impacted third molars were submitted to silver impregnation technique for quantification of argyrophilic nucleolar organizer regions (AgNOR) and immunohistochemical staining for EGFR and Ki-67. For AgNOR quantification, the mean number of active nucleolar organizer regions per nucleus (mAgNOR) and the percentage of cells with 1, 2, 3 and 4 or more AgNORs per nucleus (pAgNOR) were quantified. Ki-67 immunolabeling was quantified, whereas for EGFR, a descriptive analysis of staining patterns (membrane, cytoplasm or membrane + cytoplasm positivity) was performed. We evaluated the reduced epithelium of the enamel organ and/or islands of odontogenic epithelium present in the entire connective tissue. RESULTS: mAgNOR were 1.43 (1.0-2.42) and were significantly different among pericoronary follicles from upper and lower teeth (p = 0.041). Immunostaining of Ki-67 was negative in all cases. EGFR immunolabeling was found mainly in the cytoplasm and was more intense in islands and cords when compared to reduced epithelium of the enamel organ. CONCLUSION: Odontogenic epithelial cells of some pericoronal follicles have proliferative potential, suggesting their association with the development of odontogenic lesions. CLINICAL SIGNIFICANCE: The authors suggest that nonerupted, especially of the lower teeth, should be monitored and if necessary removed.
Assuntos
Saco Dentário/citologia , Odontogênese/fisiologia , Adolescente , Adulto , Antígenos Nucleares/análise , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Proliferação de Células , Citoplasma/ultraestrutura , Saco Dentário/ultraestrutura , Órgão do Esmalte/citologia , Órgão do Esmalte/ultraestrutura , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Receptores ErbB/análise , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Masculino , Região Organizadora do Nucléolo/ultraestrutura , Adulto JovemRESUMO
This study presents the development and evaluation of an image processing software for computer-assisted cellular structure counting. The proposed software consists of a set of processing and analytical tools which allows its use in several applications of cell and cellular structure counting. A particular application in AgNOR quantitative analysis is presented. The knowledge obtained from experienced pathologists has been codified in a sequence of processing steps in order to allow automatic estimation of the mean number of AgNORs per cell in ameloblastomas. The performance of the presented software in such application was verified by comparing the data provided by visual analysis, by two observers previously calibrated and under supervision of two experienced specialists (Group 1) and by the computer program (Group 2). No statistical difference was observed (p<0.05) between the two groups. The use of the proposed method in AgNOR applications permitted attainment of accurate and precise data without the difficulties frequently found in the traditional visual analysis method (time, training and subjectivity). The developed software is an interesting tool as an aid in the study (estimation of the number of cells and cellular structures) of malignant and benign neoplasms.
Assuntos
Ameloblastoma/ultraestrutura , Antígenos Nucleares/análise , Processamento de Imagem Assistida por Computador/métodos , Neoplasias Maxilomandibulares/ultraestrutura , Região Organizadora do Nucléolo/ultraestrutura , Patologia Bucal/instrumentação , Contagem de Células/métodos , Núcleo Celular/ultraestrutura , Proliferação de Células , Intervalos de Confiança , Humanos , Imuno-Histoquímica , Patologia Bucal/métodos , Reprodutibilidade dos Testes , SoftwareRESUMO
OBJECTIVE: Evaluate the relationships between AgNORs polymorphisms and squamous intraepithelial lesions (SIL) and squamous cell carcinoma (SCC) with HPV infection. MATERIALS AND METHODS: A study was carried out on sixty women from the state of Guerrero, Mexico. HPV detection was performed by PCR. AgNORs were identified by argentic impregnation. One hundred cells per slide were counted and classified according to the polymorphism of AgNORs dots; typical (spherical) and atypical (large, kidney-shaped and clustered). RESULTS: A total of 100% of the cases were positive for HPV infection. Nine different high-risk HPV genotypes were found, type 16 was the most common (48.6%). The AgNORs showed a significant decrease in spherical shape according to neoplastic development. The three atypical shapes showed a significant increase in SIL and SCC (p-trend<0.001). CONCLUSIONS: AgNORs polymorphism rises progressively according to the grade of histological lesions that can be useful as a prognosis for progression of SCC.
Assuntos
Carcinoma de Células Escamosas/ultraestrutura , Região Organizadora do Nucléolo/ultraestrutura , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/ultraestrutura , Cervicite Uterina/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alphapapillomavirus/classificação , Alphapapillomavirus/genética , Alphapapillomavirus/isolamento & purificação , Carcinoma de Células Escamosas/virologia , Sondas de DNA de HPV , Progressão da Doença , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Coloração pela Prata , Neoplasias do Colo do Útero/virologia , Cervicite Uterina/virologia , Adulto Jovem , Displasia do Colo do Útero/ultraestrutura , Displasia do Colo do Útero/virologiaRESUMO
OBJECTIVE: Evaluate the relationships between AgNORs polymorphisms and squamous intraepithelial lesions (SIL) and squamous cell carcinoma (SCC) with HPV infection. MATERIALS AND METHODS: A study was carried out on sixty women from the state of Guerrero, Mexico. HPV detection was performed by PCR. AgNORs were identified by argentic impregnation. One hundred cells per slide were counted and classified according to the polymorphism of AgNORs dots; typical (spherical) and atypical (large, kidney-shaped and clustered). RESULTS: A total of 100 percent of the cases were positive for HPV infection. Nine different high-risk HPV genotypes were found, type16 was the most common (48.6 percent). The AgNORs showed a significant decrease in spherical shape according to neoplastic development. The three atypical shapes showed a significant increase in SIL and SCC (p-trend<0.001). CONCLUSIONS: AgNORs polymorphism rises progressively according to the grade of histological lesions that can be useful as a prognosis for progression of SCC.
OBJETIVO: Evaluar la relación entre los polimorfismos de AgNORs con las lesiones intraepiteliales escamosas (LIE) y carcinoma de células escamosas (CCE). MATERIAL Y MÉTODOS: Se estudiaron sesenta mujeres del estado de Guerrero, México. La detección del VPH fue por PCR y los AgNORs por impregnación argéntica; se contaron 100 células y se clasificaron por tipo de polimorfismo de AgNORs: típico (esférico) y atípicos (largo, forma de riñón o de racimo). RESULTADOS: El 100 por ciento de los casos presentaron infección por VPH, se encontraron nueve genotipos diferentes de VPH de alto riesgo, el 16 fue el más común (48.6 por ciento). La forma esférica de los polimorfismos de AgNORs mostró una disminución con el desarrollo neoplásico y las atípicas incrementaron progresivamente con SIL y SCC (p-tendencia<0.001). CONCLUSIONES: Los polimorfismos AgNORs se incrementan progresivamente con el grado de lesión histológica, y pueden ser útiles en el pronóstico de progresión del carcinoma cervical.
Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Pessoa de Meia-Idade , Adulto Jovem , Carcinoma de Células Escamosas/ultraestrutura , Região Organizadora do Nucléolo/ultraestrutura , Infecções por Papillomavirus/patologia , Neoplasias do Colo do Útero/ultraestrutura , Cervicite Uterina/patologia , Alphapapillomavirus/classificação , Alphapapillomavirus/genética , Alphapapillomavirus/isolamento & purificação , Carcinoma de Células Escamosas/virologia , Displasia do Colo do Útero/ultraestrutura , Displasia do Colo do Útero/virologia , Sondas de DNA de HPV , Progressão da Doença , Genótipo , Coloração pela Prata , Neoplasias do Colo do Útero/virologia , Cervicite Uterina/virologia , Adulto JovemRESUMO
Giardia duodenalis has been described as 'anucleolated'. In this work we analysed the subcellular distribution of several nucleolar markers in Giardia nuclei using silver and immunostaining techniques for electron and confocal laser microscopy as well as expression of epitope-tagged proteins in transgenic trophozoites. We identified anteronuclear fibrogranular structures corresponding to nucleolar organising regions with recruited ribonucleoprotein complexes, rRNA and epitope-tagged fibrillarin and rRNA-pseudouridine synthase (CBF5). Recombinant fibrillarin and CBF5 were targeted to this subcompartment. This study demonstrates the presence of nucleoli in G. duodenalis and provides a model to analyse minimal requirements for nucleolar assembly and maintenance in eukaryotic cells.
Assuntos
Nucléolo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Giardia/ultraestrutura , Região Organizadora do Nucléolo/ultraestrutura , Animais , Nucléolo Celular/metabolismo , Evolução Molecular , Giardia/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica , Região Organizadora do Nucléolo/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
New karyotypic data are presented for the Astyanax fasciatus species complex from four different locations on the Upper Tibagi River in the state of Paraná, Brazil. Chromosome markers were analyzed using conventional (Ag-NOR) and molecular (FISH with 18S biotinylated probes) methods. Two cytotypes were found in cell counts with diploid number 2n=48 chromosomes and 2n=50 chromosomes, previously denominated Cytotype A and B, respectively. Two specific patterns of Ag-NORs markers (ribosomal gene activity) were found, with intra-population and inter-population variations. Cytotype A exhibited two to three chromosomes with NOR sites in the metaphases analyzed. In Cytotype B specimens, up to three markers were found, demonstrating greater intra-population and inter-population variation. All individuals with only one chromosome pair with NORs were located in the telomeric region of the short arm of Chromosome 5. This characteristic was interpreted as ancestral for the species. Another identified pattern revealed a site in the telomeric region probably in the long arm of Chromosome 4 and another submetacentric chromosome with bitelomeric marks exclusively in specimens with 2n=50 chromosomes. In the FISH analysis (ribosomal gene structure), five to seven markers were identified in Cytotype A and three to seven markers were identified in Cytotype B. Structural chromosome events and/or transposable elements are required to explain the ribosomal gene location diversity in these organisms. The results of the present study corroborate the hypothesis that the A. fasciatus of the Upper Tibagi River region constitute a species complex.