Fluorescence assay for the detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity in human blood samples / 北京大学学报(医学版)

OBJECTIVE@#To develop a simple, sensitive and robust method for rapid detection of human apurinic/apyrimidinic endonuclease 1 (APE1) in various biological samples.@*METHODS@#An abasic site-containing DNA probe with a sequence of 5'-T*T*C*C*T*C*T(ROX)AGAGXCGTT (BHQ2)C*A*C*T*G*T*AGTTTATA*C*A*G*T*GAATCTCTCTAG*T*C*T-3' ["X" represents AP site; The phosphorothioated nucleotides (at 3' side) are indicated with an asterisk after the nucleotides; ROX is 6-carboxy-X-rhodamine and BHQ2 is Black Hole quencher 2] was synthesized and used for the detection. In the presence of APE1, the DNA probe could be specifically hydrolyzed by the enzyme and release the fluorophore, resulting in strong fluorescence emission. The activity of APE1 was determined according to the rate of increase in fluorescence intensity. In this work, we modified the reaction buffer and significantly improved the performance of the method. Moreover, the method was further extended to measure the contents of APE1 in the protein extraction from peripheral blood mononuclear cells (PBMCs) extracted from human whole blood samples by density gradient centrifugation. The assay was also applied to measure the activity of APE1 in human serum samples.@*RESULTS@#With a new reaction buffer composed of 0.04% (V/V) Triton X-100, 50 mmol/L KAc, 20 mmol/L Tris-Ac, 10 mmol/L Mg(Ac)2 and 1 mmol/L dithiothreitol (DTT), the method achieved a detection limit of 0.005 U/mL (3 pg/mL) and a linear response ranging from 6 pg/mL to 1.2 ng/mL. The contents of APE1 in the protein extraction from PBMCs of eight blood samples were measured to be in the range from 0.061 to 0.40 ng/μg protein, with an average of 0.16 ng/μg protein. The recovery was 98%±5% (n=3). The levels of APE1 in the sera from 102 normal individuals (51 male and 51 female, age range: 59-75 years) were observed to be from 0.13 to 0.34 ng/mL, with a recovery of 96%±15% (n=3).@*CONCLUSION@#The new fluorescence assay was simple, rapid and sensitive, providing a practical tool to measure the activity of APE1 in serum samples and cell extracts. It also holds great potential in measurement of APE1 in many other biological samples for clinical test and laboratory research.

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.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Dynamic Regulation of APE1/Ref-1 as a Therapeutic Target Protein / 전남의대학술지

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a central role in the cellular response to DNA damage and redox regulation against oxidative stress. APE1/Ref-1 functions in the DNA base excision repair pathway, the redox regulation of several transcription factors, and the control of intracellular redox status through the inhibition of reactive oxygen species (ROS) production. APE1/Ref-1 is predominantly localized in the nucleus; however, its subcellular localization is dynamically regulated and it may be found in the mitochondria or elsewhere in the cytoplasm. Studies have identified a nuclear localization signal and a mitochondrial target sequence in APE1/Ref-1, as well as the involvement of the nuclear export system, as determinants of APE1/Ref-1 subcellular distribution. Recently, it was shown that APE1/Ref-1 is secreted in response to hyperacetylation at specific lysine residues. Additionally, post-translational modifications such as phosphorylation, S-nitrosation, and ubiquitination appear to play a role in fine-tuning the activities and subcellular localization of APE1/Ref-1. In this review, we will introduce the multifunctional role of APE1/Ref-1 and its potential usefulness as a therapeutic target in cancer and cardiovascular disease.

Dynamic Regulation of APE1/Ref-1 as a Therapeutic Target Protein / 전남의대학술지

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a central role in the cellular response to DNA damage and redox regulation against oxidative stress. APE1/Ref-1 functions in the DNA base excision repair pathway, the redox regulation of several transcription factors, and the control of intracellular redox status through the inhibition of reactive oxygen species (ROS) production. APE1/Ref-1 is predominantly localized in the nucleus; however, its subcellular localization is dynamically regulated and it may be found in the mitochondria or elsewhere in the cytoplasm. Studies have identified a nuclear localization signal and a mitochondrial target sequence in APE1/Ref-1, as well as the involvement of the nuclear export system, as determinants of APE1/Ref-1 subcellular distribution. Recently, it was shown that APE1/Ref-1 is secreted in response to hyperacetylation at specific lysine residues. Additionally, post-translational modifications such as phosphorylation, S-nitrosation, and ubiquitination appear to play a role in fine-tuning the activities and subcellular localization of APE1/Ref-1. In this review, we will introduce the multifunctional role of APE1/Ref-1 and its potential usefulness as a therapeutic target in cancer and cardiovascular disease.

Esophageal eosinophilia in pediatric patients with cerebral palsy / Eosinofilia esofágica em pacientes pediátricos com paralisia cerebral

ABSTRACT Objective: To describe the clinical picture, test results, and clinical evolution of patients with cerebral palsy associated with diagnosis of eosinophilic esophagitis, monitored at tertiary centre. Methods: Cross-sectional, retrospective and descriptive study that evaluated the medical records data of pediatric patients with diagnosis of cerebral palsy and eosinophilic esophagitis in a tertiary center of pediatric gastroenterology between August 2005 and August 2013. Results: Seven out of 131 patients with cerebral palsy had the diagnosis of eosinophilic esophagitis. The mean age at diagnosis of eosinophilic esophagitis was 52.3 months and the mean number of eosinophils in esophagus was 35 per high-power field. Symptoms more frequent were recurrent vomiting and disphagia. Endoscopic alterations found were mucosal thickening, vertical lines, mucosal opacificacion and white plaques. Conclusion: The frequency of eosinophilic esophagitis found was higher than in general pediatric population. The investigation of eosinophilic esophagitis should be done regularly in those patients, once this entity could overlap other gastrointestinal diseases. .

RESUMO Objetivo: Descrever quadro clínico, resultados dos exames e evolução clínica de pacientes com paralisia cerebral associada ao diagnóstico de esofagite eosinofílica, monitorados em um centro terciário. Métodos: Estudo transversal, retrospectivo e descritivo, que avaliou os prontuários médicos de pacientes pediátricos com diagnóstico de paralisia cerebral e esofagite eosinofílica, atendidos em um centro terciário de gastrenterologia pediátrica, entre agosto de 2005 e agosto de 2013. Resultados: Dos 131 pacientes com paralisia cerebral, 7 tiveram o diagnóstico de esofagite eosinofílica no período estudado. A idade média no momento do diagnóstico de esofagite eosinofílica foi 52,3 meses, e o número médio de eosinófilos no esôfago foi de 35 por campo de grande aumento. Os sintomas mais frequentes associados foram vômitos recorrentes e disfagia. As alterações endoscópicas encontradas foram espessamento da mucosa, linhas verticais, opacificação da mucosa e as placas esbranquiçadas. Conclusão: A frequência de esofagite eosinofílica encontrada foi maior que na população pediátrica em geral. A investigação de esofagite eosinofílica deve ser realizada regularmente nos pacientes com paralisia cerebral, pois pode haver uma sobreposição de sintomas de outras doenças gastrintestinais. .

Investigation of osteosarcoma genomics and its impact on targeted therapy: an international collaboration to conquer human osteosarcoma / 癌症

Osteosarcoma is a genetically unstable malignancy that most frequently occurs in children and young adults. The lack of progress in managing this devastating disease in the clinic has prompted international researchers to collaborate to profile key genomic alterations that define osteosarcoma. A team of researchers and clinicians from China, Finland, and the United States investigated human osteosarcoma by integrating transcriptome sequencing (RNA-seq), high-density genome-wide array comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR), Sanger sequencing, cell culture, and molecular biological approaches. Systematic analysis of genetic/genomic alterations and further functional studies have led to several important findings, including novel rearrangement hotspots, osteosarcoma-specific LRP1-SNRNP25 and KCNMB4-CCND3 fusion genes, VEGF and Wnt signaling pathway alterations, deletion of the WWOX gene, and amplification of the APEX1 and RUNX2 genes. Importantly, these genetic events associate significantly with pathogenesis, prognosis, progression, and therapeutic activity in osteosarcoma, suggesting their potential impact on improved managements of human osteosarcoma. This international initiative provides opportunities for developing new treatment modalities to conquer osteosarcoma.

Effects on survival of shRNA mediated APE/Ref1 gene silencing in rat spiral ganglion cells in oxidative stress / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of reducing APE/Ref1 expression in the cultures of rat spiral ganglion cells with oxidative damage induced by H(2)O(2).</p><p><b>METHODS</b>Primary cultured rat spiral ganglion cells were infected with small interfering RNA to APE/Ref1 (Ape1siRNA) for 72 h, followed by treating with H(2)O(2) (0, 10, 25, 50, 100 and 300 µmol/L) for 1 h , and then cultured in normal medium for 24 h. Western blot were used to detect the level of APE/Ref1 protein and phosphorylation of histone protein H2AX in the infected cells. The caspase3 activation was tested by spectrophotometric method . The cell viability was determined by MTT and the apoptosis of spiral ganglion cells was determined by terminal-deoxynucleotidyl transferase mediated nick and labeling (TUNEL).</p><p><b>RESULTS</b>Western blot showed that infection with Ape1siRNA resulted in APE/Ref1 reduced expression in the spiral ganglion cells. Exposing spiral ganglion cultures with reduced expression of APE/Ref1 to H(2)O(2) (50, 100, 300 µmol/L) for 1 h resulted in increasing in the phosphorylation of histone protein H2AX. The reduction in APE/Ref1 significantly reduced cell viability in cultures 24 h after 1 h expression to 50-300 µmol/L H(2)O(2). The apoptosis of cells and caspase 3 activity was detected significantly improved.</p><p><b>CONCLUSIONS</b>The induced of APE/Ref1 results in significantly decrease in spiral ganglion cells viability in oxidative stress. The repairing function of APE/Ref1 is necessary for optimal levels of neuronal rat spiral ganglion cells survival.</p>

Effects of polymorphisms in XRCC1 and APE1 on vinyl chloride-induced chromosome damage / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To evaluate the effects of polymorphisms in XRCC1 and APE1 genes on vinyl chloride (VC)-induced chromosomal damage in peripheral lymphocytes.</p><p><b>METHODS</b>In this study, 317 workers occupationally exposed to VC were recruited from a factory in Shandong Province, China. The micronucleus (MN) frequency in peripheral lymphocytes was used as an indicator of chromosomal damage. Polymerase chain reaction-restriction fragment length polymorphism and created restriction site combined with restriction fragment length polymorphism were used to determine the five single nucleotide polymorphisms in XRCC1 and APE1 genes in the base excision repair pathway. The association of chromosomal damage with these polymorphisms and the haplotype of XRCC1 was analyzed using Poisson regression and PHASE 2.0.2.</p><p><b>RESULTS</b>It was found that among the VC-exposed workers, individuals with XRCC1 polymorphisms (-77C/T, Arg194Trp, Arg280His, and Arg399Gln) had a significantly higher MN frequency than those with homozygous wild-type genotypes, with frequency ratios (FR) as follows, respectively: FR = 1.21, 95%CI: 1.05∼1.39 (P < 0.05); FR = 1.14, 95%CI: 1.00∼1.38 (P < 0.05); FR = 1.26, 95%CI: 1.11∼1.44 (P < 0.05); FR = 1.23, 95%CI: 1.08∼1.46 (P < 0.05). APE1 Asp148Glu was found of no significant relationship with MN frequency. Haplotype analysis of XRCC1 demonstrated that the MN frequencies in subjects with CTAA/CTAA and CCAA/CTAA were significantly higher than that in those with TCGG/TCGG (FR = 1.19, 95%CI: 1.02∼1.32, P < 0.05; FR = 1.41, 95%CI: 1.02∼1.87, P < 0.05). Furthermore, association was found between accumulated exposure to VC and XRCC1 polymorphisms (-77C/T, Arg194Trp, Arg280His, and Arg399Gln) after adjustment for age, sex, drinking, and smoking.</p><p><b>CONCLUSION</b>VC can induce chromosomal damage even when the exposure level is lower than the national occupational health standard of China (PC-TWA: 10 mg/m(3)); the polymorphisms in XRCC1 and APE1 are associated with chromosomal damage induced by VC.</p>

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.

Polymorphisms in base-excision & nucleotide-excision repair genes & prostate cancer risk in north Indian population.

Background & objectives: Genetic variation in the DNA repair genes might be associated with altered DNA repair capacities (DRC). Reduced DRC due to inherited polymorphisms may increase the susceptibility to cancers. Base excision and nucleotide excision are the two major repair pathways. We investigated the association between two base excision repair (BER) genes (APE1 exon 5, OGG1 exon 7) and two nucleotide excision repair (NER) genes (XPC PAT, XPC exon 15) with risk of prostate cancer (PCa). Methods: The study was designed with 192 histopathologically confirmed PCa patients and 224 age matched healthy controls of similar ethnicity. Genotypes were determined by amplification refractory mutation specific (ARMS) and PCR-restriction fragment length polymorphism (RFLP) methods. Results: Overall, a significant association in NER gene, XPC PAT Ins/Ins (I/I) genotype with PCa risk was observed (Adjusted OR- 2.55, 95%CI-1.22-5.33, P=0.012). XPC exon 15 variant CC genotypes presented statistically significant risk of PCa (Adjusted OR- 2.15, 95% CI-1.09-4.23, P=0.026). However, no association was observed for polymorphism with BER genes. Diplotype analysis of XPC PAT and exon 15 revealed that the frequency of the D-C and I-A diplotype was statistically significant in PCa. The variant genotypes of NER genes were also associated with high Gleason grade. Interpretation & conclusions: The results indicated that there was a significant modifying effect on the association between genotype XPC PAT and exon 15 polymorphism and PCa risk which was further confirmed by diplotype analysis of XPC PAT and exon 15 in north Indian population.

Expression of hypoxia-inducible factor 1α in osteosarcoma and its value in predicting chemosensitivity / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the prognostic values of HIF-1α, APE1, VEGF, and COX-2 protein expressions and their predictive value of tumor necrosis rate and prognosis in osteosarcoma, as well as their interrelationships.</p><p><b>METHODS</b>Formalin-fixed paraffin-embedded tissue samples were obtained from patients with osteosarcoma. Immunohistochemical assay was performed in pre-chemotherapy samples to determine the HIF-1α, VEGF, APE1, and COX-2 protein expression levels. Hematoxylin-eosin staining was used in post-operative samples to determine the tumor necrosis rate. Univariate and multivariate analyses were used to assess the impact of protein expression on prognosis.</p><p><b>RESULTS</b>Tumor tissues were obtained from 49 patients. Their median follow up was 29 months. HIF-1α was significantly correlated to every protein we tested: VEGF (P = 0.032), APE1 (P < 0.001), and COX-2 (P < 0.001). HIF-1α protein expression had a significant impact on disease free survival (P = 0.006). Expression of HIF-1α had a sensitivity of 64.7% and a specificity of 71.9% for poor pathological response (< 90% of tumor necrosis) versus good pathological response to chemotherapy (≥ 90% necrosis).</p><p><b>CONCLUSION</b>Expression of HIF-1α is a predictor of tumor response to neoadjuvant chemotherapy and outcome in osteosarcoma and is correlated with VEGF, APE1, and COX-2 expression.</p>

Anticancer clinical utility of the apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) / 癌症

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1), as a type of multifunctional protein, plays an essential role in the base excision repair (BER) pathway, which is responsible for the repair of DNA caused by oxidative and alkylation damage. As importantly, APE/Ref-1 also functions as a redox factor maintaining transcription factors in an active reduced state. APE/Ref-1 stimulates the DNA-binding activity of numerous transcription factors that are involved in cancer promotion and progression, such as AP-1 (Fos/Jun), NF-kappaB, HIF-1alpha, p53, and others. Based on the structures and functions of APE1/Ref-1, we will provide an overview of its activities and explore the budding clinical use of this protein as a target in cancer treatment, and propose that APE/Ref-1 has a great potential for application in clinical research.

Relationship between transmembrane signal transduction pathway and DNA repair and the mechanism after global cerebral ischemia-reperfusion in rats / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To investigate the protein levels of phospho-ERK and phospho-APE/Ref-1 in hippocampal neurons after global cerebral ischemia reperfusion in rats, and observe the relationship between transmembrane signal transduction and repair of DNA damage. The role of ERK signal transduction pathway following global cerebral ischemia reperfusion in rats is further discussed.</p><p><b>METHODS</b>Ninety healthy male SD rats were divided into 3 groups randomly: Sham group (S group), Ischemia reperfusion group (IR group) and Pd98059 pretreatment/ischemia reperfusion group (PD group). Global cerebral ischemia reperfusion model was established by four-vessel occlusion (4-VO) method, and reperfusion was performed 5 minutes following ischemia. Protein levels of phospho-ERK and phospho-APE/Ref-1 were detected using immunohistochemical method at 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after reperfusion, and neuron apoptosis was observed by HE and TUNEL staining.</p><p><b>RESULTS</b>In CA1 region of IR group, TUNEL positive cells began to appear at 6 h after IR, and reached the apex during 24 h to 48 h. However, TUNEL positive was most strongly exhibited in PD group. In IR group, phospho-ERK was obviously detected in CA3 region at 2 h after IR, and its level was gradually decreased from 6 h until totally absent at 48 h. Besides, phospho-ERK expression in PD group was weaker than that in IR group. For phospho-APE/Ref-1, its expression began to appear in CA1 region in IR group at 2 h after IR, with no obvious changes during 2 h to 12 h. Phospho-APE/Ref-1 expression began to decrease at 24 h and this decrease continued thereafter. Expression level of phospho-APE/Ref-1 in PD group was lower than that in IR group. Results showed the concurrence of decreased phospho-ERK expression level and increased neuron apoptosis after cerebral ischemia reperfusion, the former of which was consistent with the decrease of phospho-APE/Ref-1 expression. Also, the greater the inhibition of ERK phosphorylation was, the greater decrease of APE/Ref-1 expression occurred.</p><p><b>CONCLUSION</b>Activation of ERK signal transduction pathway increased the expression of phospho-APE/Ref-1, and thus faciliated the repair of DNA damage. So, activation of ERK signal transduction pathway may protect neurons from apoptosis after cerebral ischemia reperfusion.</p>

Expression of Ref-1 in hippocampus CA1 area of rats with Alzheimer's disease / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the expression of Redox factor-1(Ref-1) in rats with Alzheimer's disease (AD).</p><p><b>METHODS</b>AD rat model was established by a single injection of beta-amyloid peptide (Abeta25-35) into the lateral cerebral ventricle of rats. Y-shape maze task was conducted to check the ethology. At 4th, 7th and 14th day after injection, Ref-1 expression in hippocampus CA1 area was detected by immunohistochemistry.</p><p><b>RESULT</b>The numbers of repeated learning and error and the total reaction time in AD group were significantly higher than those in control group at 7th and 14th day after injection of Abeta25-35 (P<0.05). At 4th day after injection of Abeta25-35, the Ref-1 expression was increased with time prolongation in AD group(P<0.01).</p><p><b>CONCLUSION</b>Injection of Abeta25-35 in lateral cerebral ventricle can decrease the learning and memory abilities of rats, and results also indicate that Ref-1 expression in hippocampus may be involved in the development of AD.</p>

Adenoviral-mediated APE/Ref-1 expression protects rat spiral ganglion cells from oxidative damage / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To address the question if apurinic/apyrimidinic endonuclease/redox factor 1 (APE/Ref-1) involved in preventing spiral ganglion cells oxidative damage after oxidative stress.</p><p><b>METHODS</b>Primary cultured rat spiral ganglion cells were infected with the adenovirus containing APE/Ref-1 for 48 h, then treated with H2O2 (0, 10, 25, 50, 100, 300 micromol/L) for 1 h, and finally changed back into normal medium. Western blot were used to detect the level of APE/Ref-1 protein in the infected cells to ensure APE/Ref-1 over expression as a result of adenovirus infection. The cell viability was determined by MTT and the apoptosis of spiral ganglion cells was determined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL).</p><p><b>RESULTS</b>Western blot showed that infection of adenovirus resulted in APE/Ref-1 over expression in the spiral ganglion cells. Over expression of APE/Ref-1 significantly improved cell viability in cultures treated with different concentration H2O2 from 50 to 300 micromol/L However, the apoptosis of cells was significantly inhibited.</p><p><b>CONCLUSIONS</b>Over expression of APE/Ref-1 could protect spiral ganglion cells from oxidative damage.</p>

Relationship of genetic polymorphism in APE1 and ADPRT to risks of chronic benzene poisoning / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the relationship between genetic polymorphisms in apurinic/apyrimidinic endonuclease (APE1) and ADP ribosyltransferase (ADPRT) and individuals' susceptibility to chronic benzene poison ing (BP).</p><p><b>METHODS</b>A case-control study was conducted. One hundred and fifty-two B P patients and 152 workers occupationally exposed to benzene without poisoning manifestations were investigated. The mismatched bases combined to create restriction site with restrained fragment length polymorphism technique (CRS-RFLP) was used for detecting the single nucleotide polymorphisms (SNPs) at Asp148Glu of APE1 gene and Val762Ala of ADPRT gene.</p><p><b>RESULTS</b>There was no significant difference in the distribution of genotypes of APE1Asp148Glu and ADPRTVal762Ala between the patients and the control groups. Compared with individuals having genotype of APE1Asp148Glu T/T without habit of alcohol consumption, there was a 4.13 times increased risk of BP for the alcohol user with genotype of APE1Asp148Glu T/T (OR = 4.13, 95% CI: 1.07 - 15.85, P = 0.03). The analysis of Logistic regression showed that smoking may play some role in modifying the risk of cironic benzene poisoning (OR = 0.33, 95% CI: 0.14 - 0.75, P = 0.01).</p><p><b>CONCLUSION</b>The genetic polymorphisms in APE1Asp148Glu, ADPRTVal762Ala are not related to the risk of BP. Potential interaction is found between alcohol consumption and polymorphism of APE1Asp148Glu. Further study is needed to elucidate this interaction.</p>

Construction and identification of a multiple myeloma-specific APE1 siRNA expression vector / 中华血液学杂志

<p><b>OBJECTIVE</b>To construct a multiple myeloma (MM)-specific APE1siRNA expression vector, and detect the specific knock-down effect of the siRNA on expression of APE1 protein.</p><p><b>METHODS</b>APE1siRNA cDNA sequence was designed, synthesized and inserted into pSilencer 2.0-U6 linear expression vector. pSilencer APE1siRNA was digested by enzyme EcoRI and BamHI, then linear vector and IgP fragments were conjugated by T4 DNA ligase. pSilencer IgP-APE1siRNA and pSilencer IE-IgP-APE1siRNA were digested by enzyme EcoRI or XhoI. Linear vector and IE or Kappa fragments were conjugated by T4 DNA ligase. Then a MM specific pSilencer K-IE-IgP-APE1siRNA was cloned. The recombinant products were identified by DNA sequencing and enzyme digestions at each step. pSilencer K-IE-IgP-APE1siRNA plasmid was transfected to KM3, HOS, MDA-231 cells by liposome. APE1 gene silence induced by RNAi was analysed by Western blot.</p><p><b>RESULTS</b>APE1 protein in KM3 cells could be knocked down effectively and specifically by pSilencer K-IE-IgP-APE1siRNA vector. After 2 days, the level of APE1 protein in KM3 cells transfected with siRNA was 0.118 +/- 0.047, while that transfected with plasmid only was 0.988 +/- 0.029. The efficiency of gene silence was 90%.</p><p><b>CONCLUSION</b>A MM specific APE1siRNA expression vector was successfully constructed.</p>

Study on significance of single nucleotide polymorphisms in the redox domain of aprimidinic/apurinic endonuclease/redox factor-1 gene in Chinese sporadic colorectal cancer / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the single nucleotide polymorphisms(SNPs) in the redox domain of aprimidinic/apurinic endonuclease/redox factor-1(APEX) gene and the development of sporadic colorectal cancer.</p><p><b>METHODS</b>One hundred and fifty cases of sporadic colorectal cancers and 143 peripheral blood samples from healthy population were screened for genetic polymorphisms or mutations in the redox domain by denaturing gradient gel electrophoresis followed by DNA sequencing.</p><p><b>RESULTS</b>There were two SNPs identified in the redox domain of APEX gene, namely, 453G to T and 1247A to G. The gene frequencies of 453T and 1247G were 1.3% and 5.7%, respectively, in patient group, while 1.05% and 4.55%, respectively, in healthy population. The genotype distribution at the two sites in healthy population was consistent with Hardy-Weinberg equilibrium. There was no difference in gene frequencies at the two sites between cancer patients and healthy population.</p><p><b>CONCLUSION</b>The polymorphisms in the redox domain of APEX gene are irrelevant to the development of sporadic colorectal cancer, but their distribution may vary greatly among tribes.</p>

Site-Specific Mutagenesis in Escherichia coli by Bulky Exocyclic Amino-Substituted Guanine and Adenine Derivatives in Double-Stranded or Gapped Plasmids / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: 7-Bromomethylbenz[alpha]anthracene is a known mutagen and carcinogen. The mutagenic potency of its two major DNA adducts, i.e., N2-(benz[alpha]anthracen-7-ylmethyl)-2'-deoxyguanosine (b[alpha]a2G) and N6-(benz[alpha]anthracen-7-ylmethyl)-2'-deoxyadenosine (b[alpha]a6A), as well as the simpler benzylated analogs, N2-benzyl-2'-deoxyguanosine (bn2G) and N6-benzyl-2'-deoxyadenosine (bn6A), were determined in E. coli. MATERIALS AND METHODS: Double-stranded and gapped plasmid vectors were used to determine the mutagenicity of b[alpha]a2G, b[alpha]a6A, bn2G and bn6A in E. coli. The four, suitably protected, bulky exocyclic amino-substituted adducts were incorporated into 16-base oligodeoxyribonucleotides, in place of normal guanine or adenine residues, which form part of the ATG initiation codon for the lacZ' alpha-complementation gene. The site-specifically modified oligodeoxyribonucleotides were then incorporated into double-stranded plasmids, which contained uracil residues in the complementary strand in the vicinity of the initiation codon. The uracil residues lead to the creation of a gap in the complementary strand due to the actions of E. coli uracil-DNA glycosylase and AP endonuclease. Following the transfection of these plasmid vectors into E. coli strain GP102, a lacZ alpha complementing version of the parent strain AB1157, their propensity to induce mutation was investigated. RESULTS: The percentages of mutant colonies produced by the four modified nucleosides, in both the double-stranded and gapped plasmid vectors, were not significantly different from those produced by the unmodified plasmids. The mutagenicities of the b[alpha]a2G and b[alpha]a6A were extremely low, and a totally unexpected result, whereas, those of the bn2G and bn6A were undetectable. CONCLUSION: In this E. coli site-specific mutagenesis system, these bulky aralkylated adducts exhibited no significant mutagenicities, either with or without SOS induction.