Análise da imunoexpressão de proteínas de reparo do DNA envolvidas nas vias BER e NER em lesões odontogênicas epiteliais benignas / Immunoexpression analysis of DNA repair proteins involved in BER and NER pathways in benign epithelial odontogenic lesions

As lesões odontogênicas epiteliais benignas apresentam comportamento biológico heterogêneo e patogênese ainda não totalmente esclarecida. As vias de reparo do ácido desoxirribonucleico (DNA) atuam em tipos específicos de danos ao material genético, realizando o reparo e regulando diversos processos celulares. Dentre as principais vias de reparo do DNA, destacamse o reparo por excisão de bases (BER) e o reparo por excisão de nucleotídeos (NER). Investigações têm demonstrado que as proteínas envolvidas nessas vias se encontram desreguladas e, por vezes, altamente expressas em algumas neoplasias malignas, contribuindo para a progressão tumoral. Levando em consideração a heterogeneidade do comportamento biológico das lesões odontogênicas epiteliais benignas e a escassez de estudos que tenham avaliado a expressão de proteínas de reparo do DNA nestas lesões, este trabalho avaliou a imunoexpressão de proteínas da via BER (APE-1 e XRCC-1) e NER (XPF) em ameloblastomas (AMEs) sólidos (n = 30), ceratocistos odontogênicos não sindrômicos (CONS) (n = 30), ceratocistos odontogênicos sindrômicos (COS) (associados à Síndrome de Gorlin) (n = 29), cistos dentígeros (CDs) (n = 30) e folículos dentários (FDs) (n = 20). A análise da expressão imunoistoquímica de APE-1, XRCC-1 e XPF foi realizada de forma quantitativa por um avaliador previamente calibrado e sem acesso aos dados clínicos dos casos. Em cinco campos de maior imunorreatividade, foram quantificadas as células positivas e negativas para as proteínas no componente epitelial de todos os casos, sendo estabelecido o percentual de células positivas em relação ao número total de células contadas para cada anticorpo. As marcações nucleares e citoplasmáticas foram analisadas separadamente para APE-1 e XPF, enquanto apenas a imunoexpressão nuclear foi considerada para XRCC-1. As comparações das medianas dos percentuais de imunorreatividade em relação aos grupos estudados foram realizadas por meio dos testes não paramétricos de Kruskal-Wallis e Mann-Whitney. Possíveis correlações entre a expressão de APE-1, XRCC-1 e XPF foram avaliadas por meio do teste de correlação de Spearman. O nível de significância foi estabelecido em 5% (p < 0,05). Foi verificada uma maior imunoexpressão nuclear de APE-1 nos CONSs, COSs e AMEs sólidos, em comparação com os CDs (p < 0,001). Dentre todos os grupos avaliados, a expressão citoplasmática de APE1 só foi encontrada em 4 CONSs e 6 COSs. A expressão nuclear de XRCC-1 foi estatisticamente maior nos CONSs e COSs em relação aos CDs (p < 0,05). Em nível nuclear, a expressão de XPF foi significativamente maior nos CONSs e COSs em relação aos CDs e AMEs (p < 0,05) e, embora sem significância estatística, foi observada uma maior expressão nuclear dessa proteína nos AMEs quando comparado aos CDs. Em relação à expressão citoplasmática de XPF, foi observada uma maior expressão nos COSs em relação aos CDs (p = 0,04). Nenhuma diferença estatisticamente significativa foi encontrada entre as expressões nucleares de APE-1, XRCC-1 e XPF entre CONSs e COSs (p > 0,05). Além disso, todas as lesões odontogênicas estudadas revelaram uma maior expressão estatisticamente significativa de APE-1 (nuclear), XRCC-1 (nuclear) e XPF (nuclear e citoplasmática) quando comparados aos FDs (p < 0,05). Para todas as lesões, o teste de correlação de Spearman mostrou uma correlação positiva entre a expressão nuclear de APE-1 e XRCC-1 ou XPF, em nível nuclear (p < 0,05). Os resultados deste estudo sugerem um potencial envolvimento das proteínas APE-1, XRCC-1 e XPF na patogênese das lesões odontogênicas epiteliais benignas, com destaque para aquelas com comportamento biológico mais agressivo (AU).

The benign epithelial odontogenic lesions present a heterogeneous biological behavior and their pathogenesis are not fully understood. The deoxyribonucleic acid (DNA) repair pathways act on specific types of damage to the genetic material, performing the repair and regulating several cellular processes. Among the main DNA repair pathways, the most notable are the base excision repair (BER) and the nucleotide excision repair (NER). Investigations have shown that the proteins involved in these pathways are deregulated and sometimes highly expressed in some malignancies, contributing to tumor progression. Taking into account the heterogeneity of the biological behavior of benign epithelial odontogenic lesions and the scarcity of studies that have evaluated the expression of DNA repair proteins in these lesions, this study evaluated the immunoexpression of BER (APE-1 and XRCC-1) proteins and NER (XPF) in solid ameloblastomas (AMEs) (n = 30), non-syndromic odontogenic keratocysts (NSOKCs) (n = 30), syndromic odontogenic keratocysts (SKOCs) (associated with Gorlin's Syndrome) (n = 29), dentigerous cysts (DCs) (n = 30) and dental follicles (DFs) (n = 20). The immunohistochemical analysis of APE-1, XRCC-1 and XPF was performed quantitatively by a previously calibrated evaluator and without access to the clinical data of the cases. In five fields of higher immunoreactivity, positive and negative cells were quantified for the proteins in the epithelial component of all cases, and the percentage of positive cells was established in relation to the total number of cells counted for each antibody. Nuclear and cytoplasmic markers were analyzed separately for APE-1 and XPF, while only nuclear immunoexpression was considered for XRCC-1. The comparisons of the median percentages of immunoreactivity in relation to the studied groups were performed using the non-parametric Kruskal-Wallis and MannWhitney tests. Possible correlations between the expression of APE-1, XRCC-1 and XPF were assessed by Spearman's correlation test. The level of significance was set at 5% (p < 0.05). A higher nuclear immunoexpression of APE-1 in the NSOKCs, SOKCs and solid AMEs was verified in comparison with the DCs (p < 0.001). Among all the evaluated groups, the cytoplasmic expression of APE-1 was only found in 4 NSOKCs and 6 SOKCs. Nuclear expression of XRCC-1 was statistically higher in NSOKCs and SOKCs than in DCs (p < 0.05). At the nuclear level, XPF expression was significantly higher in NSOKCs and SOKCs than in DCs and AMEs (p < 0.05) and, although without statistical significance, a higher nuclear expression of this protein was observed in AMEs when compared to CDs. Regarding the cytoplasmic expression of XPF, a greater expression was observed in the SOKCs in relation to the DCs (p = 0.04). No statistically significant difference was found between the nuclear expressions of APE-1, XRCC-1 and XPF between NSOKCs and SOKCs (p > 0.05). In addition, all the odontogenic lesions studied revealed a statistically significant expression of APE-1 (nuclear), XRCC-1 (nuclear) and XPF (nuclear and cytoplasmic) when compared to DFs (p < 0.05). For all lesions, Spearman's correlation test showed a positive correlation between nuclear expression of APE-1 and XRCC-1 or XPF at the nuclear level (p < 0.05). The results of this study suggest a potential involvement of APE-1, XRCC-1 and XPF proteins in the pathogenesis of benign epithelial odontogenic lesions. The role played by these proteins may be more important in odontogenic lesions with more aggressive biological behavior (AU).

Correlations between Ape1/Ref-1, ICAM-1 and IL-17A Levels in Serum and Radiation Pneumonitis for Local Advanced Non-small Cell Lung Cancer Patients / 中国肺癌杂志

BACKGROUND@#The main manifestations of radiation pneumonitis are injury of alveolar epithelial and endothelial cells, abnormal expression of cytokines, abnormal proliferation of fibroblasts and synthesis of fibrous matrix. The occurrence of radiation pneumonitis is associated with multiplecytokine level abnormality. These cytokines can also be used as bio-markers to predict the occurrence of radiation pneumonitis. This study was to evaluate the correlation between the change of apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1), intercellular adhesion molecules 1 (ICAM-1) and interleukin-17A (IL-17A) before and after radiotherapy and radiation pneumonitis for local advanced non-small cell lung cancer (NSCLC) patients with concurrent chemoradiotherapy.@*METHODS@#NSCLC patients (68 cases) were treated with concurrent radiotherapy and chemotherapy, every patient's normal tissue were controlled with a same radation dose. 68 local advanced NSCLC patients with concurrent chemoradiotherapy were detected the levels of Ape1/Ref-1, ICAM-1 and IL-17A in serum by ELISA before radiotherapy and in the 14th week after radiotherapy. Acute and advanced radiation pulmonary injury was graded according to Radiation Therapy Oncology Group/European Organization For Research and Treatment (RTOG/EORTC) diagnostic and grading criteria. Grade 2 or more radiation pneumonitis was taken as the main end point.@*RESULTS@#Eighteen cases out of 68 developed radiation pneumonitis, 50 of 68 cases have no radiation pneumonia development. There was no significant change of Ape1/Ref-1 levels before and after radiotherapy in radiation pneumonitis group (P>0.05). There was no significant change of Ape1/Ref-1 concentration in serum after radiotherapy between radiation pneumonitis group and non-radiation pneumonitis group (P>0.05). Compared with before radiotherapy, upregulation degree of ICAM-1 levels in radiation pneumonitis group was significantly higher than that in non- radiation pneumonitis group (P<0.05). There was no significant change of IL-17A concentration before and after radiotherapy in radiation pneumonitis group, but after radiotherapy IL-17A concentration in serum were remarkably higher than that in non-radiation pneumonitis group (P<0.05). Correlation analysis found that the change of ICAM-1 before and after radiotherapy has no obvious correlation with the incidence of radiation pneumonitis, and IL-17A change has obvious correlation with the incidence of radiation pneumonitis.@*CONCLUSIONS@#On the basis of strictly controlling radiation dose on normal tissue, IL-17A in serum could be the predictive factors of radiation pneumonitis for local advanced NSCLC patients with concurrent chemoradiotherapy.

Identification of New Potential APE1 Inhibitors by Pharmacophore Modeling and Molecular Docking

Apurinic/apyrimidinic endonuclease 1 (APE1) is an enzyme responsible for the initial step in the base excision repair pathway and is known to be a potential drug target for treating cancers, because its expression is associated with resistance to DNA-damaging anticancer agents. Although several inhibitors already have been identified, the identification of novel kinds of potential inhibitors of APE1 could provide a seed for the development of improved anticancer drugs. For this purpose, we first classified known inhibitors of APE1. According to the classification, we constructed two distinct pharmacophore models. We screened more than 3 million lead-like compounds using the pharmacophores. Hits that fulfilled the features of the pharmacophore models were identified. In addition to the pharmacophore screen, we carried out molecular docking to prioritize hits. Based on these processes, we ultimately identified 1,338 potential inhibitors of APE1 with predicted binding affinities to the enzyme.

Accelerated processing of solitary and clustered abasic site DNA damage lesions by APE1 in the presence of aqueous extract of Ganoderma lucidum.

The stimulatory effect of the aqueous extract of G. lucidum, a basidiomycetes class fungus in the APE1-enzyme-mediated processing of solitary and bistranded clustered abasic sites DNA damages is presented. Abasic sites are considered the most common type of DNA damage lesions. Our study shows enhanced activity of APE1 in the processing of abasic sites in the presence of the polysaccharides fraction of G. lucidum. Remarkable increase in the amount of single-strand breaks (SSBs) and double-strand breaks (DSBs) from solitary and bistranded clustered abasic sites respectively with APE1 in the presence of the extract was found. This trend is maintained when abasic sites in DNA oligomers are exposed to fibroblast cell extracts in the presence of the extract. While DNA conformational alteration is negligible, APE1 enzyme shows characteristic changes in the alpha helix and beta strand ratio after incubation with G. lucidum extract. The enhanced reactivity of APE1 at the molecular level in the presence of G. lucidium is attributed to this effect. This study potentially amplifies the scope of the use of G. lucidum, which was earlier shown to have only reactive oxygen species (ROS) scavenging properties with regards to DNA damage inhibition.

Altered Secretory Activity of APE1/Ref-1 D148E Variants Identified in Human Patients With Bladder Cancer / 대한배뇨장애요실금학회지

PURPOSE: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox modulation. Recently, serum and urinary APE1/Ref-1 levels were reported to be increased in patients with bladder cancer. Genetic variations of APE/Ref-1 are associated with the risk of cancer. However, the effect of APE1/Ref-1 variants on its secretory activity is yet unknown. METHODS: APE1/Ref-1 variants were evaluated by DNA sequencing analysis of reverse transcription polymerase chain reaction products in coding DNA sequences (CDS) of APE1/Ref-1 in bladder tissue samples from patients with bladder cancer (n=10). Secretory activity of APE1/Ref-1 variants was evaluated with immunoblot and enzyme-linked immunosorbent assay of the culture medium supernatants. RESULTS: Four different substitution mutants (D148E, I64V/D148E, W67R/D148E, and E86G/D148E) of APE1/Ref-1 were identified in bladder cancer specimens. However, deletion mutants of APE1/Ref-1 CDS were not found. The secretory activity of the APE1/Ref-1 variants (D148E, I64V/D148E, and E86G/D148E) was increased compared to that of wild type APE1/Ref-1. Furthermore, the secretory activity in basal or hyperacetylated conditions was much higher than that in APE1/Ref-1 D148E-transfected HEK293 cells. CONCLUSIONS: Taken together, our data suggest that the increased secretory activity of D148E might contribute to increased serum levels of APE1/Ref-1 in patients with bladder cancer.

Smile Design: From Ape To Human.

Abstracts: Background & Objective: The domain of periodontics has changed from being strictly a health service to one, where smile enhancement has been brought to the forefront of treatment planning. In the composition of a beautiful smile, the form, balance, symmetry and relationship of the elements make it attractive or unattractive. Altered passive eruption is one of the conditions where a large portion of the anatomic crown remains covered by the gingiva which compromises dentofacial aesthetics. Periodontal plastic procedures, such as the basic gingivectomy, osseous correction or the apically positioned flap, may be used to change the silhouette form of teeth and their relative proportion. Here is a case of a 22-year-old healthy male with altered passive eruption (type 1A) with treatment that resulted in a revised silhouette form for the tooth which is more elliptical and attractive so as to resolve the unwarranted excessive display of gingiva apparent during smiling.

Cytoprotective effect of rhamnetin on miconazole-induced H9c2 cell damage

BACKGROUND/OBJECTIVES: Reactive oxygen species (ROS) formation is closely related to miconazole-induced heart dysfunction. Although rhamnetin has antioxidant effects, it remained unknown whether it can protect against miconazole-induced cardiomyocyte apoptosis. Thus, we investigated the effects of rhamnetin on miconazole-stimulated H9c2 cell apoptosis. MATERIALS/METHODS: Cell morphology was observed by inverted microscope and cell viability was determined using a WelCount(TM) cell proliferation assay kit. Miconazole-induced ROS production was evaluated by fluorescence-activated cell sorting with 6-carboxy-2',7'-dichlorofluoroscein diacetate (H2DCF-DA) stain. Immunoblot analysis was used to determine apurinic/apyrimidinic endonuclease 1 (APE/Ref-1) and cleaved cysteine-aspartic protease (caspase) 3 expression. NADPH oxidase levels were measured using real-time polymerase chain reaction. RESULTS: Miconazole (3 and 10 microM) induced abnormal morphological changes and cell death in H9c2 cells. Rhamnetin enhanced the viability of miconazole (3 microM)-treated cells in a dose-dependent manner. Rhamnetin (1 and 3 microM) treatment downregulated cleaved caspase 3 and upregulated APE/Ref-1 expression in miconazole-stimulated cells. Additionally, rhamnetin significantly reduced ROS generation. CONCLUSIONS: Our data suggest that rhamnetin may have cytoprotective effects in miconazole-stimulated H9c2 cardiomyocytes via ROS inhibition. This effect most likely occurs through the upregulation of APE/Ref-1 and attenuation of hydrogen peroxide levels.

Gemcitabine-resistant Human Pancreatic Cancer Cell Line Overexpresses APE1/Ref-1 / 胃肠病学

Background:Gemcitabine is the main drug for chemotherapy of advanced pancreatic cancer,however,the prognosis of pancreatic cancer patients has not been changed obviously because of the high innate and acquired resistance of cancer cells to gemcitabine. Aims:To investigate the correlation of DNA repair and expression of human APE1 / Ref-1(apurinic/apyrimidinic endonuclease 1 / redox factor-1),the key enzyme in base excision repair pathway,with the resistance of pancreatic cancer to gemcitabine. Methods:A gemcitabine-resistant human pancreatic cancer cell line SW1990-0. 5 with a resistance index of 9. 32 and its parental cell line SW1990 were treated with gemcitabine. DNA injury was assessed by comet assay. Expressions of APE1 / Ref-1 mRNA and protein were determined by real-time PCR and Western blotting, respectively. Results:In comet assay,after treated with gemcitabine for 24 hours,OTM value of SW1990-0. 5 and SW1990 cells were 0. 32 ± 0. 13 and 26. 96 ± 6. 83,respectively. Expression level of APE1 / Ref-1 mRNA in SW1990-0. 5 cells was 2. 48 ± 0. 49;and expression levels of APE1 / Ref-1 protein in SW1990-0. 5 and SW1990 cells were 1. 57 ± 0. 08 and 0. 84 ± 0. 06,respectively. Statistically significant differences were existed in all these parameters between SW1990-0. 5 and SW1990 cells(P all < 0. 05). Conclusions:DNA repair might be correlated with the resistance of pancreatic cancer to gemcitabine,and up-regulation of APE1 / Ref-1 might contribute to this resistance by its function on DNA repair.

APE1/Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by endogenous and exogenous agents. APE1 acts as a reductive activator of many transcription factors (TFs) and has also been named redox effector factor 1, Ref-1. For example, APE1 activates activator protein-1, nuclear factor kappa B, hypoxia-inducible factor 1alpha, paired box gene 8, signal transducer activator of transcription 3 and p53, which are involved in apoptosis, inflammation, angiogenesis and survival pathways. APE1/Ref-1 maintains cellular homeostasis (redox) via the activation of TFs that regulate various physiological processes and that crosstalk with redox balancing agents (for example, thioredoxin, catalase and superoxide dismutase) by controlling levels of reactive oxygen and nitrogen species. The efficiency of APE1/Ref-1's function(s) depends on pairwise interaction with participant protein(s), the functions regulated by APE1/Ref-1 include the BER pathway, TFs, energy metabolism, cytoskeletal elements and stress-dependent responses. Thus, APE1/Ref-1 acts as a 'hub-protein' that controls pathways that are important for cell survival. In this review, we will discuss APE1/Ref-1's versatile nature in various human etiologies, including neurodegeneration, cancer, cardiovascular and other diseases that have been linked with alterations in the expression, subcellular localization and activities of APE/Ref-1. APE1/Ref-1 can be targeted for therapeutic intervention using natural plant products that modulate the expression and functions of APE1/Ref-1. In addition, studies focusing on translational applications based on APE1/Ref-1-mediated therapeutic interventions are discussed.

Utilidad del APE libre en el diagnóstico de cáncer de próstata / Use of free PSA ratio in the diagnosis of prostate cancer

El Cáncer de próstata (CaP) constituye una importante causa de muerte en nuestro país. La razón Antígeno Prostático Específico libre/ Antígeno Prostático Específico total (APE-L/T) es una de las medidas descritas para mejorar la pesquisa de la enfermedad. MATERIAL Y MÉTODOS: Se realizó un estudio retrospectivo analítico en el Hospital Naval de Viña del Mar entre enero 2007 a diciembre 2011 que incluyó 588 pacientes con biopsias prostáticas que presentaban APE- T entre 2-10 ng/ml. Se evaluó la edad al diagnóstico, el valor de APE-T y la razón APE-L/T en relación al resultado histológico de las biopsias. Se realizaron curvas de rendimiento diagnóstico (ROC) para APE- T y para la razón APE-L/T. Se calculó sensibilidad y especificidad para diferentes puntos de corte para la razón APE-L/T. RESULTADOS: 33 por ciento de las biopsias fueron positivas para CaP. Los valores de la razón APE-L/T fueron significativamente más bajos en pacientes con CaP (p<0.001), alcanzando un área bajo la curva ROC 0,615. El mejor punto de corte para la razón APE-L/T fue de 15 por ciento con una sensibilidad de 60 por ciento y una especificidad de 58 por ciento. Para la razón APE-L/T > 25 por ciento la sensibilidad es 20 por ciento y la especificidad 91 por ciento. En cambio, el APE-T no mostró diferencias estadísticamente significativas (p=0,1) con un AUC 0,55. CONCLUSIONES: El APE-T por sí solo no parece tener capacidad discriminante para detectar CaP cuando los valores se encuentran entre 2-10 ng/ml. La razón APE-L/T tiene una utilidad limitada frente al paciente para decidir efectuar o no una biopsia de próstata.

Prostate cancer (PCa) is one of the major causes of death in our country. The Free/Total Prostate specific antigen ratio (f/t PSA) is one of the measurements that have been used to improve the diagnosis of this disease. MATERIAL AND METHODS: A retrospective analytic study in the Almirante Nef Hospital in Viña del Mar between January 2007 and December 2011 was made, which included 588 patients with prostate biopsies that had tPSA between 2-10 ng/ml. The age at diagnosis was evaluated and the value of tPSA and the f/t PSA ratio were compared with the histological results of the biopsies. Curves were performed for the diagnostic yield (ROC) for tPSA and the f/t PSA ratio. The sensitivity and specificity for different cut-off points for the f/t PSA ratio in the diagnosis of PCa were also calculated. RESULTS: 33 percent of the biopsies were positive for PCa. The f/t PSA ratio values were significantly lower in patients with CaP (p<0.001), reaching an area under the curve of 0,615. The best cutoff for f/t PSA ratio was 15 percent with 60 percent sensitivity and 58 percent specificity. For f/t PSA ratio > 25 percent sensitivity was 20 percent and specificity was 91 percent. PSA showed no statistically significant differences (p = 0.1) with AUC: 0.55. CONCLUSIONS: tPSA alone does not seem to have discriminatory power to detect PCa in patients when values are between 2-10 ng/ml. The f/t PSA ratio has a limited utility to decide to perform a prostate biopsy or not.

Genetic similarity between APEC and Escherichia coli strains isolated from Guaruba guarouba in a survey on healthy captive psittacine birds / Similaridade genética entre APEC e cepas de Escherichia coli isoladas de Guaruba guarouba em um estudo com psitacídeos hígidos de cativeiro

The role of psittacine birds as a reservoir of avian pathogenic Escherichia coli (APEC) is not known but would be helpful in understanding the human – animal interface, since the enteric microbiota of these birds consists of Gram positive bacteria. The aim of this study was to identify the presence of APEC in feces of clinically healthy Guaruba guarouba. To do this, we isolated and analyzed E. coli from cloacal fecal samples taken from 87 psittacine birds from six zoologic parks, three commercial breeders and one conservation breeder. Of the 87 birds examined, 46 (52.87%) presented E. coli in feces. The presence of the following eight virulence genes was determined by the polymerase chain reaction (PCR): irp2, iucD, iss, vat, cvi/cva, tsh, astA, and papC, and 29 (63.04%) of 46 E. coli isolates tested were positive at least one of the eight genes studied. The frequency of virulence genes observed in isolates of E. coli were 32.6% (15/46) irp2, 26% (12/46) iucD, 19.5% iss (9/46), 17.4% vat (8/46), 17.4% cvi/cva (8/46), 8.7% tsh (4/46), 4.4% astA (2/46) and 0% papC (0/46). The isolates were grouped in 13 genotypic profiles according to virulence gene combinations, but only 2 isolates were classified as APEC, with the pattern iuc, iss, cvi/cva, irp + and iuc, iss, cvi/cva, irp, tsh, vat +. This study reveals the presence of APEC in clinically healthy captive G. guarouba, suggesting that these psittacine birds may act as reservoir for pathogenic microorganisms. Epidemiological studies are needed to determine the relevance of this species as a reservoir and the implications for conservation of endangered species G. guarouba.

O papel dos psitacídeos como reservatório de Escherichia coli patogênicas para aves (APEC) não é conhecido, mas será útil para a compreensão da interface humano-animal, uma vez que a microbiota entérica destas aves é composta por bactérias Gram-positivas. O objetivo deste estudo foi identificar a presença de APEC em fezes de Guaruba guarouba clinicamente saudáveis. Para isso, foram isoladas e analisadas E. coli presentes em fezes cloacais coletadas de 87 psitacídeos, alojados em seis zoológicos, três criatórios comerciais e um criatório conservacionista. Das 87 aves examinadas, 46 (52,87%) apresentaram E. coli nas fezes. A presença de oito genes de virulência foi determinada pela reação em cadeia pela polimerase (PCR): irp2, iucD, iss, vat, cvi/cva, tsh, astA, e papC, e 29 (63,04%) dos 46 isolados foram positivos para pelo menos um dos oito genes estudados. A frequência dos genes de virulência observada nos isolados de E. coli foi 32.6% (15/46) irp2, 26% (12/46) iucD, 19.5% iss (9/46), 17.4% vat (8/46), 17.4% cvi/cva (8/46), 8.7% tsh (4/46), 4.4% astA (2/46) e 0% papC (0/46). Os isolados foram agrupados em 13 perfis genotípicos de acordo com combinações de genes de virulência, mas apenas duas amostras foram classificadas como APEC, com o perfil iuc, iss, cvi/cva, irp + e iuc, iss, cvi/cva, irp, tsh, iuc, iss, +. Este estudo revela a presença de APEC em aves de cativeiro (G. guarouba) clinicamente saudáveis, sugerindo que estes psitacídeos possam atuar como reservatórios de micro-organismos patogênicos. Estudos epidemiológicos são necessários para determinar a relevância desta espécie como reservatório e as implicações para a conservação da espécie ameaçada G. guarouba.

Expression of DNA Repairing Enzymes in the Cerebral Tissue of the Rat Fetus After Hypoxic Injury / 체질인류학회지

Hypoxia is one of the major causes of neonatal mortality. Hypoxia-induced tissue injuries are resulted from complex mechanisms such as DNA damage and apoptosis. In this study, we aimed to elucidate the changes in the expression of DNA repairing enzymes such as 8-hydroxyguanine glycosylase 1 (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1) and brain derived neurotrophic factor (BDNF) in the fetal cerebral tissue after intrauterine hypoxic injury. For this study, pregnant Sprague-Dawley rats were exposed to hypoxic gas (10% O2, 5% CO2, 85% N2) for 2 or 4 hours at postconception day 14.5 and 15.5. After 24 hours, the animals were anesthetized with ethyl ether and fetuses were obtained by laparatomy. Hematoxylin-eosin stain, immunohistochemical stain, and western blot were employed for analysis. The caspase-3 immunolabeled cells were significantly increased within the cerebral cortex after hypoxic injury. The expressions of OGG1, APE1, and BDNF were also increased in the cerebral tissue after hypoxic injury at post-conception day 14.5, in a dose-dependent manner. However, the expression of BDNF was significantly decreased in the cortical tissue exposed to hypoxic injury at postconception day 15.5. These results demonstrate that fetal hypoxic injury induces apoptosis of the nerve cells and promotes the expressions of the DNA repairing enzymes and neurotrophic factors. In addition, these results suggest that protection mechanisms against hypoxic injury alter along the progression of the fetal development.

Relación entre el diagnóstico clínico preoperatorio y el histopatológico posoperatorio, en pacientes con cáncer prostático localizado / Relationship between clinical preoperative diagnosis and postoperative histology in patients with localized prostate cancer

Introducción: Las variables relevantes preoperatorias con que cuenta el urólogo para una toma de decisión frente a un cáncer prostático localizado son: la edad, el tacto rectal, el antígeno prostático específico (APE) e informe histológico de la biopsia por punción con el Gleason. Además se pueden incluir otras variables como el volumen prostático, número de muestras de biopsias positivas, porcentaje de la muestra comprometida, etc. Nosotros quisimos evaluar el grado de concordancia entre el diagnóstico clínico-patológico preoperatorio con el hallazgo histológico, posoperatorio en pacientes prostatectomizados, debido a la implicancia pronóstica y en la toma de decisión que pudiese tener. Material y Método: Se estudiaron retrospectivamente 119 prostatectomías radicales entre marzo de 2004 y junio de 2009. Se consideraron: edad, tacto, antígeno prostático específico (APE) y score de Gleason. Se excluyeron pacientes con tratamiento antiandrogénico u hormonal neoadjuvante. Resultados: En el preoperatorio la mediana de edad fue de 66 años (61-68), de APE 7,35 ng/ml (5,38-11,8) y de Gleason fue de 6 (5-7). El 87,4 por ciento de los pacientes tenía un APE >4,0 ng/ml. El 54 por ciento (n= 64) tenía un estadio clínico T1c y el 46 por ciento (n= 55) un estadio T2. En el posoperatorio 23,5 por ciento (n= 28) tuvo un estadio pT2 y el 74 por ciento (n= 88) un estadio pT3. En pacientes con estadio pT2 el APE preoperatorio fue de 5,9 ng/ml (4,4-9,4), en el estadio pT3 fue de 7,9 ng/ml (5,7-12,8). El score de Gleason en pT2 fue de 5 (5-6), en el pT3 fue de 6 (5-7). No encontramos diferencia de edad en los estadios pT2 (67 años) y pT3 (68 años). Conclusiones: En el estudio histopatológico posoperatorio de pacientes con estadio clínico T1c y T2, se confirmó un estadio pT2 sólo en 23,5 por ciento, el 74 por ciento tenían un estadio pT3 (a, b). En el cáncer prostático localizado, el tacto rectal no fue útil en su correlación con el estadio histológico...

Introduction: Relevant preoperative variables in patients with localized prostate cancer are: age, digital rectal examination (DRE), prostatic specific antigen (PSA) level and Gleason score in the transrectal biopsy. Other variables include prostate volume, number of positive biopsy samples, percentage of involvement in the biopsy, etc. We evaluated the agreement between the preoperative clinico pathologic diagnosis and the postoperative histology report in patients submitted to prostatectomy. Material and method: This is a retrospective review of 119 radical prostatectomies performed between March 2004 and June 2009. We recorded age, DRE, PSA level, and Gleason score. Patients receiving anti-androgenic treatment or neoadjuvant hormonal treatment were excluded. Results: Preoperative findings: median age was 66 years (61-68), median PSA level was 7.35 ng/ml(5.38-11.8) and median Gleason score was 6 (5-7). PSA level >4 ng/ml was found in 87.4 percent of the patients. Clinical stage T1c was found in 54 percent (n=64) of the cases whereas 46 percent (n=55) were stage T2. Postoperative findings: stage pT2 was found in 23.5 percent (n=28) of the patients whereas 74 percent (n =88)were pT3 stage. In pT2 patients, preoperative PSA was 5,9 ng/ml (4.4-9.4). In pT3 patients, PSA was7.9 ng/ml (5.7-12.8). Gleason score in pT2 was 5 (5-6); in pT3 patients, Gleason score was 6 (5-7). No age difference was found between pT2 stage (67 years) and pT3 stage (68 years).Conclusions: Postoperative histology in patients with T1c and T2 stages confirmed a pT2 stage only in 23.5 percent of the cases; 74 percent of the cases were pT3 (a,b) stage. In localized prostate cancer, DRE was not useful for the correlation with pathologic staging, especially for stage pT3 cases. Preoperative Gleason score was relatively useful; we found understaging 36.2 percent of the cases and overstaging 21.8 percent of the patients. These variables should be considered in the initial evaluation of...

Clinical application of invasive ventilation in treatment of acute pulmonary edema and prognosis analysis / 中华急诊医学杂志

Objective To observe the efficacy of invasive ventilation (Ⅳ) in treatment of acute pulmonary edema (APE), and to explore the factors associated with prognosis. Method From March 2005 to December 2007, 23 APE patients, who were hospitalized in the EICU of People' s Hospital of Peking University and the con-ventional treatment and noninvasive ventilation were not effective, were treated by Ⅳ (PEEP 5～18 cmH2O). Blood pressure, heart rate, respiratory rate, and arterial blood gas values were recorded accurately before and after ventilation and compared with each other. Regression analysis was used to analyze the factors associated with prog-nosis. Resets Three hours after Ⅳ, the blood pressure, heart rate, respiratory rate, arterial blood gas were sig-nificanfly improved (P <0.01). Among the 23 patients, 11 survived, and the other 12 patients died. Nine pa-tients died of MOF. Among 16 patients with cardiac function Ⅲ-Ⅳ, 10 died. Among 15 patients with acute my-ocardial infarction, 9 died. Among 11 patients with renal insufficiency, 9 died. The multivariate logistic regression analysis showed that the reduced left ventricular ejection fraction, the lower mean arterial pressure, and the in-creased creatinine were the independent predictor of prognosis. Conclusions Invasive ventilation is an effective method of treating patients with acute pulmonary edema. Proper ventilation may improve the cardiac function and clinical symptoms, but it's not the fundamental measure for increasing cure rate. 1he renal insufficiency and heart failure are the independent predictor of prognosis.

Ape1/Ref-1 Stimulates GDNF/GFR alpha1-mediated Downstream Signaling and Neuroblastoma Proliferation

We previously reported that glial cell line-derived neurotropic factor (GDNF) receptor alpha1 (GFR alpha1) is a direct target of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1). In the present study, we further analyzed the physiological roles of Ape1/Ref-1-induced GFRalpha1 expression in Neuro2a mouse neuroblastoma cells. Ape1/Ref-1 expression caused the clustering of GFR alpha1 immunoreactivity in lipid rafts in response to GDNF. We also found that Ret, a downstream target of GFRalpha1, was functionally activated by GDNF in Ape1/Ref-1-expressing cells. Moreover, GDNF promoted the proliferation of Ape1/Ref-1-expressing Neuro2a cells. Furthermore, GFR alpha1-specific RNA experiments demonstrated that the downregulation of GFR alpha1 by siRNA in Ape1/Ref-1-expressing cells impaired the ability of GDNF to phosphorylate Akt and PLC gamma-1 and to stimulate cellular proliferation. These results show an association between Ape1/Ref-1 and GDNF/GFR alpha signaling, and suggest a potential molecular mechanism for the involvement of Ape1/Ref-1 in neuronal proliferation.

El uso de la detección de células prostáticas en la circulación sanguínea podría disminuir el número de biopsias prostáticas necesarias para detectar cáncer prostático / Detection of circulating prostate cells could avoid prostate biopsies

Introducción: El uso del antígeno prostático específico como pesquisa para cáncer prostático ha significado según algunos estudios una disminución en la mortalidad y un cambio hacia las etapas más precoces. Pero, implica también que aproximadamente70 por ciento de los hombres con un APE elevado tendrán una biopsia negativa para cáncer, asumiéndose los riesgos de hemorragia e infección del procedimiento. Presentamos un estudio sobre la detección de células prostáticas en la circulación sanguínea como examen complementario y los resultados de la biopsia prostática. Método y pacientes: A hombres que cumplían con los criterios para someterse a una biopsia prostática, se les tomó una muestra de sangre. Las células mononucleares fueron separadas usando centrifugación diferencial y las células prostáticas detectadas usando anticuerpos monoclonales contra el APE y identificadas con inmunocitoquímica. Los resultados de la presencia o ausencia de las CPCs fueron comparados con los resultados de la biopsia. La biopsia fue dirigida por ecografía y tomada siguiendo la norma estándar en sextante. Resultados: Participaron 358 hombres, de éstos, 91 pacientes cumplieron con los criterios para una biopsia, de los cuales 86 se les tomó una biopsia. La ausencia de CPCs fue asociada con una biopsia negativa en 94,7 por ciento (54/57) y hubo CPCs detectadas en 24/27 (89 por ciento) de los casos con una biopsia positiva para cáncer. En 3 casos biopsia positiva CPC negativa el cáncer fue de bajo grado y localizado. Hubo una sensibilidad de 91,5 por ciento y una especificad de 89,0 por ciento. Conclusiones: Hombres negativo para CPCs tienen una alta posibilidad de una biopsia prostática negativa (94,7 por ciento), en estos hombres es posible postergar la biopsia con un monitoreo cuidadoso del APE sérico. Evitando biopsias no necesarias disminuirán los riesgos asociados al paciente sin aumentar los riesgos de no detectar un cáncer agresivo.

Introduction: The widespread use of PSA screening for prostate cancer has decreased mortality and increased early stage detection. However, approximately 70 percent of biopsies will be negative in men with an increased PSA, incurring in the associated risks of haemorrhage and infection. We report the use of circulating prostate cells (CPCs) as a complementary test and compare the results with the associated prostate biopsy. Patients and Methods: Men fulfilling biopsy criteria had a blood sample taken, the mononuclear cells were separated using differential centrifugation and detected using monoclonal antibodies against PSA and identified using monoclonal antibodies against PSA and identified using immunocytochemistry. Standard ultrasound guided sextant biopsy was used. The presence or absence of CPCs was compared with prostate biopsy results. Results: Of 358 men participating in the study 91 fulfilled biopsy criteria of which 86underwent biopsy. The absence of CPCs was seen in 94.7 percent of cases (54/57) with a negative biopsy. In the 3 remaining cases the CPC negative patient had a low grade local cancer. Overall there was a sensitivity of 91.5 percent and specificity of 89.0 percent. Conclusions: Men with negative CPC have a high probability of a negative biopsy, in these patients the biopsy could be deferred with close monitoring of PSA levels, thus avoiding biopsy complications. This would avoid unnecessary risks without jeopardizing early stage cancer detection.

Construction of thioredoxin-DsRed and APE/ref-1-EGFP fusion protein vectors and their localization in 293T cells / 基础医学与临床

Objective To construct thioredoxin and APE/ref-1 fusion protein expression vector and to investigate their subcellular localization of the fusion proteins in 293Tcells.Methods The APE/ref-1 cDNA was cloned by RT-PCR from PC12 cell.APE/ref-1-EGFP fusion protein expression vector was constructed through subcloning.Thioredoxin cDNA was subcloned into pDSred1-1 from pQE30-TRX plasmid by PCR,and thioredoxin-DsRed fusion protein expression vector was constructed in pCMV5 plasmid.The expression and subcellular localization of the fusion proteins in 293T cells transfected with the vectors by calcium phosphate was analyzed by fluorescent microscopy.Results The results demonstrated that thioredoxin——DsRed and APE/ref-1-EGFP fusion protein expression vectors were successfully constructed and expressed in 293T cells.APE/ref-1-EGFP fusion protein was located only in nucleus,and thioredoxin-DsRed fusion protein was located in cytoplasm as well as nucleus.ConclusionThis study has set up a solid base for further to study on the dynamic interaction between thioredoxin and APE/ref-1 fusion proteins.

Rapid Loss of Apurinic/Apyrimidinic Endonuclease and Subsequent Apoptosis in Kainate-Induced Seizure Model / 대한간질학회지

PURPOSE: The DNA repair enzyme, apurinic/apyrimidinic endonuclease (APE) plays a role in base excision repair pathway involved in repairing apurinic/apyrimidinic (AP) site after oxidative stress. To reveal the relationship between APE and neuronal apoptosis associated with oxidative stress after kainate treatment, the temporal change of APE expression was investigated in kainate-induced seizure model. METHODS: Status epilepticus was induced by unilateral intrahippocampal injection of kainate. Superoxide anion radical production and DNA oxidation were evaluated by in situ detection of oxidized hydroethidine and 8-hydroxyguanine (8-OHG) immunore activity. APE expression was examined by Western blot and immunohistochemical analysis. DNA fragmentation was visualized with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. RESULTS: Cell loss occurred at 24 hr in CA1, CA2, and CA3 after kainate-injection. 8-OHG immunoreactivity and oxidized hydroethidine were increased comparing with control after kainate-injection. APE immunoreactivity was decreased 4 and 24 hours in the hippocampus after kainate-injection. TUNEL-positive cells were observed 24 hours but not 4 hours in hippocampus after kainate-injection. In double labeling with APE and TUNEL, TUNEL-positive cells did not show APE immunoreactivity. These data showed that cellular oxidative stress was increased, thereby APE was decreased in the hippocampus after kainate-injection. Also, it was shown that the reduction of APE preceded DNA fragmentation. CONCLUSION: This study suggests that rapid loss of APE may produce the failure of DNA repair-machinary and then induce neuronal apoptosis following kainate-injection.

Antitumor effect of GALV membrane fusion glycoprotein on lung adenocarcinoma in vivo and in vitro / 第三军医大学学报

Objective To investigate the antitumor effect of special promoter-controlled Gibbon ape leukemia virus membrane fusion glycoprotein (GALV.fus) mediated by type Ⅰ herpes simplex virus (HSV-Ⅰ) on lung adenocarcinoma. Methods Recombinant HSV-Ⅰ plasmids encoding GALV.fus was introduced into green monkey kidney cells(Vero)by liposome to amplify the virus, and then the virus was transfected into lung adenocarcinoma (A549), human fetal fibroblasts (HFL-Ⅰ GNHu 5) and human lung adenocarcinoma xenografts which were established in nude mice subcutaneously to observe antitumor and cytotoxic effect in vitro and in vivo; Recombined cytomegalovirus (CMV) containing GALV.fus or enhanced green fluorescence protein were served as control. Results Recombinant HSV-Ⅰ virus were packed successfully. Heterotransplantative tumourigenicity of the tumour was 100% in nude mice after A549 cells were inoculated. Recombinant HSV-Ⅰvirus exerted obvious antitumor effects in vitro, with relative survival rate of 23%, while for CMV virus containing GALV. fus, the rate was 20%, and for CMV virus encoding EGFP, the rate was 68%. Recombinant HSV-Ⅰvirus also showed striking antitumor effect on the implanted tumor. Conclusion GALV.fus has powerful effect against lung cancer in vitro and in vivo and maybe a promising candidate for gene therapy.

In vitro study of the treatment of human osteosarcoma by pSilence APE1 combined with neutron-ray / 解放军医学杂志

Objective Apuinic/apyrimidic endonuclease/redox effector factor-1(APE1/Ref-1,abbreviated as APE1) is a major member of the base excision repair(BER) pathway involved in oxidative DNA damage repair.To knock down APE1 gene expression in HOS cells with pSilence APE1,and explore its effect in combination with 252Cf neutron ray radiotherapy.Methods The constructed APE1 siRNA expression vector pSilence APE1 was transfected into HOS cells by SuperFect Transfection liposome,and it was used to knock down the expression of APE1.The HOS cells and transfected HOS cells were respectively irradiated by 252Cf neutron ray,then the cell survival(D0),DNA single strand breaks(SSB) and cell apoptosis were determined by clone formation assay,alkaline comet assay and flow cytometer.Results The cell-survival curve was plotted by clone formation assay,the D0 value was 2.80 vs.1.89 and Dq value was 2.66 vs.2.00 for the control and transfected HOS cells,respectively,after being irradiated by 252Cf neutron ray.The tail moments at 2,5 and 10 Gy were 6.664?0.648 vs.7.997?0.542,20.322?1.433 vs.25.238?1.185 and 33.909?1.245 vs.39.191?1.052,respectively,for the control and transfected HOS cells,and the cell apoptosis rate at 2,5 and 10 Gy was 4.00 vs.5.68,5.91 vs.7.55 and 9.63 vs.13.51,respectively,for the control and transfected HOS cells.All these findings showed significant difference between the two groups(P