Apurinic/apyrimidinic endonuclease 1 mRNA level in peripheral blood neutrophils is associated with asthma / El nivel de ARNm de la endonucleasa 1 apurínica/apirimidínica en los neutrófilos de sangre periférica se asocia con el asma

Abstract Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional key protein. Recent studies suggest APE1 is closely associated with inflammatory response, but its role in asthma remains unknown. We recruited 116 patients with asthma, including 50 with severe asthma (NSA) and 66 with non-severe asthma (SA), and 140 controls. Serum APE1 was detected using the ELISA method. APE1 mRNA in peripheral blood neutrophils and eosinophils were detected using real-time PCR assays. Compared to healthy controls, we observed significant elevations of serum APE1 mRNA levels in peripheral neutrophils (~1.75 folds increase, p<0.05) and eosinophils (~2.2 folds increase, p<0.05) in patients with asthma. The peripheral blood neutrophil APE1 mRNA can distinguish asthmatic patients from healthy controls with the area under the curve (AUC) 0.893 and a 95% confidence interval (CI) 0.847-0.938 (p < 0.001). Also the APE1 mRNA can identify severe asthma from non-severe asthma (AUC 0.759, 95% CI, 0.674-0.846; p < 0.001). However, The serum APE1 and eosinophil mRNA levels did not correlate with asthma incidence and severity. Our finding confirms the association between APE1 and asthma and suggests that peripheral blood neutrophil APE1 mRNA may be used as a marker for this condition.

Resumen La endonucleasa apurínica/apirimidínica 1 (APE1) es una proteína clave multifuncional. Estudios recientes sugieren que APE1 está estrechamente asociada con la respuesta inflamatoria, pero hasta el momento se desconoce su papel en el asma. Reclutamos a 116 pacientes con asma, incluidos 50 con asma grave (NSA) y 66 con asma no grave (SA), y 140 controles. Se detectó APE1 en suero usando el método ELISA. El ARNm de APE1 en neutrófilos y eosinófilos de sangre periférica se detectó mediante ensayos de PCR en tiempo real. En comparación con los controles sanos, observamos una elevación significativa de los niveles séricos de ARNm de APE1 en pacientes con asma en neutrófilos periféricos (aumento de ~1,75 veces, p<0,05) y eosinófilos (aumento de ~2,2 veces, p<0,05). El ARNm de APE1 de neutrófilos de sangre periférica puede distinguir a los pacientes asmáticos de los controles sanos con un área bajo la curva (AUC) de 0,893 y un intervalo de confianza (IC) del 95% de 0,847 a 0,938 (p<0,001). Además, el ARNm de APE1 puede identificar el asma grave del asma no grave (AUC 0,759, IC del 95%, 0,674-0,846; p < 0,001). Sin embargo, el nivel sérico de APE1 y ARNm de eosinófilos no mostró correlación con la incidencia y la gravedad del asma. Nuestro hallazgo confirma la asociación entre APE1 y asma y sugiere que el ARNm de APE1 de neutrófilos en sangre periférica puede usarse como marcador para el asma.

Relationship between the G894T polymorphism of endothelial nitric oxide synthase gene and lipid metabolism among hypertensive disorder complicating pregnancy patients / 解剖学报

Objective To investigate the relationship between the G894T polymorphism of the endothelial nitric oxide synthase (eNOS) gene and the lipid metabolism in patients with irypertensive disorder complicating pregnancy (HDCP). Methods The 528 cases of HDCP patients admitted to the Xingtai Third Hospital from January 2016 to January 2020 were selected as the research objects, and 128 normal pregnant women during the same period were selected as the control group. The fasting peripheral venous blood of all stud)' subjects in the early morning was collected, and blood lipid indexes, cystatin C (CysC) and uric acid levels and other biochemical index levels were measured. According to the blood lipid level, it was divided into normal blood lipid group and dyslipidemia group. The dyslipidemia group included 4 subgroups [ hyper triglyceride (TG) blood group (T G

Sensitive detection of alkaline phosphatase based on terminal deoxynucleotidyl transferase and endonuclease Ⅳ-assisted exponential signal amplification / 药物分析学报

Alkaline phosphatase(ALP)is widely expressed in human tissues.ALP plays an important role in the dephosphorylation of proteins and nucleic acids.Therefore,quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods.Terminal deoxynucleotidyl transferase(TdT)catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3'-OH end of single-stranded DNA in the absence of a template.In this study,we developed a highly sensitive and selective method based on TdT and endonuclease Ⅳ(Endo Ⅳ)to quantify ALP activity.After ALP hydrolyzes the 3'-PO4 end of the substrate and generates 3'-OH,TdT can effectively elongate the 3'-OH end with deoxynucleotide adenine triphosphate(dATP)and produce a poly A tail,which can be detected by the poly T probes.Endo Ⅳ digests the AP site in poly T probes to generate a fluorescent signal and a new 3'-OH end,leading to the generation of exponential fluorescence signal amplification.The substrate for TdT elongation was optimized,and a limit of detection of 4.3×10-3 U/L was achieved for ALP by the optimized substrate structure.This method can also detect ALP in the cell lysate of a single cell.This work has potential applications in disease diagnosis and biomedical detection.

Effect of acupuncture on neurological function, cerebral infarction volume, thrombolysis time window and cerebral cell apoptosis signaling pathway in cerebral infarction rats / 针刺研究

OBJECTIVE: To observe the effect of acupuncture (Acupunct) on cerebral infarction volume and expression of poly ADP ribose polymerase 1 (PARP1), apoptosis-inducing factor (AIF) and endonuclease G (Endo-G) in the cerebral cortex tissue at different time-points after cerebral ischemia (CI) in acute cerebral infarction rats, so as to explore its underlying mechanisms in prolonging time window of thrombolysis. METHODS: Forty-eight SD rats were randomly divided into sham operation, model, intravenous thrombolysis (IVT)-4.5 h, IVT-6 h, IVT-9 h, Acupunct＋IVT-4.5 h, Acupunct ＋IVT-6 h, Acupunct＋IVT-9 h groups (n＝6 in each group). The CI model was established by using modified autologous thromboembolism via the right common carotid artery. Two hours after modeling, rats of the Acupunct groups received Acupunct stimulation of "Shuigou" (GV26) and bilateral "Neiguan" (PC6) for 30 min. Thrombolysis was conducted by injection of recombinant human tissue-type plasminogen activator (rt-PA, 10 mg/kg) via caudal vein. The neurological deficit was assessed with reference to Bederson's methods. 2，3，5-triphenyltetrazolium chloride (TTC) staining was used to assess the cerebral infarction volume, and the expression of cerebral PARP1, AIF and Endo-G proteins detected by Western blot. RESULTS: Compared with the sham operation group, the neurological score and percentage of cerebral infarction volume, expression levels of PARP1, AIF and Endo-G proteins were significantly increased in the model group (P0.05). The levels of neurological score, percentage of cerebral infarction volume, and AIF expression were significantly lower in both the Acupunct＋IVT 4.5 h and Acupunct＋IVT-6 h groups than in the simple IVT-4.5 h and simple IVT-6 h groups, respectively (P<0.05), and the expression levels of PARP1 and Endo-G proteins were obviously lower in the Acupunct＋IVT-4.5 h group than in the IVT-4.5 h group (P<0.05). Endo-G proteins were obviously lower in the Acupunct＋IVT-9 h group than in the IVT-9 h group (P<0.05). CONCLUSION: Acupuncture may improve neurological function, reduce cerebral infarction volume and prolong the time window of thrombolysis in CI rats, which may be associated with its effect in suppressing AIF/PARP1/ Endo-G signaling.

FEN1 expression in oral carcinoma-associated fibroblasts and its relationship with PCNA / 口腔疾病防治

Objective@#To research the expression levels of FEN1 and PCNA in carcinoma-associated fibroblasts (CAFs) and analyze their correlation. @*Methods@#Fresh specimens of oral squamous cell carcinoma tissues and normal oral mucosal tissues excised during oral and maxillofacial plastic surgery were collected. Primary oral CAFs and normal fibroblasts (NFs) were obtained by tissue culture, identified by immunocytochemistry and divided into the CAF and NF groups. Western blot and quantitative real-time PCR were used to detect the protein and mRNA expression levels of both FEN1 and PCNA in the oral CAFs and NFs. The correlation between FEN1 and PCNA expression in oral CAFs was analyzed. @*Results@#Oral CAFs and oral NFs were successfully cultured and identified from 12 samples. Both the protein and mRNA expression levels of FEN1 and PCNA were higher in the oral CAFs than NFs, but there were no significant differences (P > 0.05). In the oral CAFs, the linear correlation coefficient between FEN1 and PCNA was 0.677 (P = 0.016) at the mRNA level, indicating a strong positive correlation; however, at the protein level, no correlation was found (P > 0.05). @*Conclusion@# In primary cultured oral CAFs and NFs, there were no significant differences in the FEN1 and PCNA protein and mRNA expression levels. However, in the CAFs, the mRNA levels of FEN1 and PCNA had a strong positive correlation. The relationship and the regulatory mechanism of the two genes require further study.

Associations of NEIL3 rs12645561 with severity of coronary atherosclerosis in Chinese Han population of Henan and Hubei regions / 临床检验杂志

Objective To assess the association of polymorphism at rs12645561 locus of Nei endonuclease Ⅷ-like 3 ( NEIL3) gene with the severity of coronary atherosclerosis in Han population of Hubei and Henan regions in central China. Methods In 947 cases undergoing coronary angiography, the severity of coronary atherosclerosis was calculated by lesion vessel number scores and Gensini scores.The genotypes of rs12645561 were analyzed by high resolution melting curve. The plasma NEIL3 levels were measured by en-zyme-linked immunosorbent assay. Results The minor allele T of rs12645561 was significantly associated with the risk of coronary ar-tery disease (χ2=12.165, P<0.05) including increased lesion vessel scores and Gensini scores (χ2=14.745 and 15.615,P<0.05). The variant risk genotypes ( CC+CT) of rs12645561, body mass index of more than 25 kg/m2 , hypelipidemia and smoking habit were all the independent risk factors for higher Gensini scores ( OR=1.50, 1.54, 2.01 and 1.42, respectively, P<0.05) . There were signifi-cantly inverse correlations of plasma NEIL3 levels with the distribution of rs12645561, lesion vessel number scores and Gensini scores ( P<0.05) . Conclusion rs12645561 may correlate with the severity of coronary atherosclerosis, and contribute to the development of coronary atherosclerosis of Han population of Henan and Hubei regions. rs12645561 may also affect the levels of NEIL3 protein.

Two-plasmid scarless genetic modification in Escherichia coli hfq and rne-710 / 生物工程学报

Gene modification is an important technique to understand gene function. We firstly constructed Δhfq::Spe and Δrne-710::Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.

Therapeutic oligonucleotides: a review / 生物工程学报

Oligonucleotides are widely used as effective tools to regulate gene expression and drugs for targeted gene therapy. Therefore, they are potentially useful for the treatment of viral, tumor and hereditary diseases. Therapeutic oligonucleotides include antisense oligonucleotide, small interference RNA (siRNA), Ribozyme, DNAzyme, anti-gene, CpG, decoy and aptamer. Therapeutic oligonucleotides usually carry certain modifications, such as phosphorothioates, fluoro or locked nucleic acids, to enhance the stability and specificity, and reduce the side-effects, because natural oligonucleotides have poor stability in vivo, low specificity and side effects. Now oligonucleotides are usually manufactured by chemical synthesis, with low purity and high cost. Here, we review a novel thermocyclic reaction for the amplification of oligonucleotides, referred to as Polymerase-endonuclease Amplification Reaction (PEAR) catalyzed by two thermostable enzymes. PEAR is simple, efficient, and stable. Comparing with traditional chemical synthesis, PEAR-based enzymatic production of oligonucleotides could be a robust alternative method for the large-scale production of therapeutic or non-therapeutic oligonucleotides.

Expression and clinical significance of MMR protein and MLH1 promoter methylation testing in endometrial cancer / 中华妇产科杂志

Objective To explore the expression and clinical significance of mismatch repair (MMR)protein and MLH1 promoter methylation testing in endometrial cancer(EC). Methods A total of 420 cases with EC diagnosed by the surgical pathology examination from the Department of Pathology of PLA General Hospital, MLH1,MSH2,MSH6 and PMS2 protein in EC were detected by immunohistochemistry and methylation-specific multiplex ligation-dependent probe amplification(MS-MLPA) testing. Results (1)Of the 420 tumor cases, the total expression loss rate of MMR protein was 34.5%(145/420), the expression loss rates of MLH1,MSH2,MSH6 and PMS2 protein were respectively 17.1%(72/420), 8.1% (34/420), 7.4%(31/420), 26.2%(110/420)and loss rates of MLH1 and PMS2,MSH2 and MSH6 were 16.7%(70/420), 6.2%(26/420). When there was a loss of MMR protein expression, any one or more protein expression deletions in MLH1, PMS2, MSH2 and MSH6, it could be Lynch syndrome related endometrial carcinoma(LS-EC). The expression loss rate of MMR protein in the poorly differentiated endometrioid adenocarcinoma was higher than that in the well differentiated endometrioid adenocarcinoma(P<0.05).(2) The expression loss rate of MMR and PMS2 protein had statistically significant between the endometrioid adenocarcinoma and non-endometrioid adenocarcinoma(P<0.01). The expression loss rate of MSH2 protein had statistically significant in the stage Ⅲ(P<0.01). Moreover, there were also significant differences in depth of myometrial invasion and lymph node metastasis between the expression loss rate of MMR protein (P<0.05).(3)The expression loss rate of MLH1 protein was 72 cases and 57 cases had MLH1 promoter methylation testing(excluding those who were not qualified for DNA testing). The positive rate was 47.4% (27/57). Therefore, these patients were sporadic endometrial cancer, not non-LS-EC. Conclusions MMR protein may be play an important role in the development of endometrial cancer and be indicated poor prognosis. Immunohistochemical staining and MLH1 promoter methylation detection may be play an important role in the screening of the LS-EC.

Detection of Low-abundance Point Mutations by Competitive Strand Assisted Endonuclease Ⅳ Signal Amplification System / 华中科技大学学报（医学）（英德文版）

Genetic mutations are important molecular biomarkers for cancer diagnosis and surveillance.Therefore,the development of methods for mutation detection characterized with straightforward,highly specific and sensitive to low-level mutations within various sequence contexts is extremely needed.Although some of the currently available methods have shown very encouraging results,their discrimination efficiency is still very low.Herein,we demonstrate a fluorescent probe coupled with blocker and property of melting temperature discrimination,which is able to identify the presence of known or unknown single-base variations at abundances down to 0.1％ within 20 min.The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 10.15-38.48.The method is sequence independent,which assures a wide range of application.The new method would be an ideal choice for high-throughput in vitro diagnosis and precise clinical treatment.

Expression of endonuclease G in the brain tissue of rats with chronic fluorosis / 中华地方病学杂志

Objective To investigate the possible mechanism of endonuclease G (Endo G)-mediated non-Caspase-dependent apoptotic pathway in brain neuronal apoptosis in chronic fluorosis rats.Methods Sixty Sprague Dawley (SD) rats (half male and half female) were randomly divided into two groups:control group fed with tap water with fluoride content ＜ 0.5 mg/L and fluorine group in which sodium fluoride was added into drinking water with fluoride content of 50.0 mg/L.Both groups were fed with standard food with fluorine content ＜ 0.5 mg/kg.The experiment period was 10 months.At the end of the experiment,all the animals were sacrificed,and brain tissue was taken.Flow cytometry was used to examine apoptosis rate,immune-histochemistry was employed to detect the distribution of Endo G in brain tissue;Western blotting was used to test the protein expression of Endo G.Results Compared to the apoptosis rate of control group [(1.3 ± 0.6)％,(1.9 ± 0.3)％],the apoptosis rate in hippocampus and cortex of rats with chronic fluorosis [(2.6 ± 0.6)％,(3.1-± 0.7)％] was significantly increased (t =3.1,3.4,all P ＜ 0.05).The Endo G positive neurons and their degree of staining in CA1,CA2,CA3 and CA4 of hippocampus,frontal cortex as well as the upper layer of parietal cortex [(11.1 ± 2.2),(10.2 ± 1.9),(9.8 ± 3.1),(9.9 ± 1.6),(10.6 ± 2.9),(8.2 ± 2.4),(11.1 ± 2.8) scores] in rats with chronic fluorosis were significantly higher than those in the control group [(5.8 ± 1.8),(6.7 ± 2.6),(5.2 ± 2.4),(7.2 ± 2.1),(7.7 ± 2.6),(6.1 ± 1.9),(8.1 ± 2.6) scores,t =2.9,2.5,2.4,2.3,2.2,2.5,2.3,P ＜ 0.01 or ＜ 0.05].The protein level of Endo G in the mitochondria of rat brains with chronic fluorosis [(86.4 ± 7.2)％,(83.9 ± 6.8)％] was significantly lower than that of control group [(100.0 ± 6.1)％,(100.0 ± 5.5)％,t =2.6,2.3,all P ＜ 0.05].Meanwhile,the protein level of Endo G in the nucleus of neurons from chronic fluorosis rats [(117.5 ± 6.4)％,(115.2 ± 6.2)％] was significantly higher than that of the control [(100.0 ± 5.2)％,(100.0 ± 5.5)％,t =2.5,2.2,all P ＜ 0.05].Conclusion The high expression of Endo G and nuclear transfer are related to the neuron apoptosis in chronic fluorosis rat,which may be one of the mechanisms of brain injury of the disease.

Production of Restriction Endonuclease Not Ⅰ Utilizing CpG DNA Methylase M.Sss Ⅰ Co-expression Vector / 中国生物工程杂志

Restriction endonucleases are important molecular biology tools for DNA recombination.Because of the cleavage of DNA,their recombinant expression is difficult with low yields and complicated purification processes.In commercial productions,the technology that uses specific methylases to protect host DNA from digestion of the expressed restriction enzymes was cumbersome and practically limited.For solving this problem the expression of restriction enzyme Not Ⅰ was performed by using the DNA methylase M.Sss Ⅰ derived from Spiroplasma sp.MQ1 which specifically kept CpG sequence methylated.The methylated DNA was protected from the cutting of Not Ⅰ whose recognition sequence contained CpG.The gene of methylase M.Sss Ⅰ was introduced into Escherichia coli ER2566 and constitutively expressed,resulting in the CpG methylation pattern of the host DNA.Restriction enzyme Not Ⅰ was successfully expressed in this E.coli strain.Furthermore,by adding a purification tag to one terminus of the enzyme,recombinant Not Ⅰ was prepared as a highly purified and active product through two simple Ni-affinity and anion exchange chromatography steps.This expression system can be applied for the preparation of a series of restriction enzymes with CpG in their recognition sequences.

Value of endonuclease domain containing 1 in progression of prostate cancer / 中国病理生理杂志

AIM: To analyze the difference of endonuclease domain containing 1 (ENDOD1) expression be-tween benign prostatic hyperplasia ( BPH ) tissues and prostate cancer ( PCa ) tissues and to investigate the effect of ENDOD1 on the biological function of human prostate cancer cells .METHODS: The BPH samples ( n=20 ) and PCa samples (n=21) were processed and analyzed according to the instruction of immunohistochemical (IHC) staining.The mRNA and protein levels of ENDOD 1 in the normal prostate epithelial cells and prostate cancer cells were evaluated by RT -qPCR and Western blot , respectively .The recombinant plasmids pCMV-N-Flag-ENDOD1 was constructed and was trans-fected into the human prostate cancer cells .The proliferation , apoptosis , migration and invasion abilities of the prostate cancer cells were evaluated by MTT assay , flow cytometry, Transwell migration and Matrigel invasion assays , respectively. RESULTS:The analysis of variance of the immunoreactivity score showed that PCa tissues with high Gleason score dis -played significantly lower ENDOD1expression than that with low Gleason score and BPH (P<0.05).The expression of ENDOD1 at mRNA and protein levels in PC3 cells and DU145 cells was significantly lower than that in the LNCap cells (P<0.05).The proliferation of DU145 transfected with ENDOD1 was inhibited.The flow cytometry indicated that ENDOD1 over-expression in the DU145 cells resulted in a notable increase in G0/G1 phase arrest (P<0.05), but the ap-optotic rates showed no statistical difference .The results of Transwell assay showed that migration and invasion abilities of the cells were also inhibited after transfection with over-expressing ENDOD1 plasmid (P<0.05).CONCLUSION: The expression of ENDOD1 significantly decreased in prostate cancer with high Gleaon score .Meanwhile, the ENDOD1 is spe-cifically down-regulated in androgen independent prostate cancer (AIPC) cell lines.Over-expression of ENDOD1 remark-ably inhibits the proliferation , migration and invasion abilities of AIPC .

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.

A putative Type IIS restriction endonuclease GeoICI from Geobacillus sp. – A robust, thermostable alternative to mezophilic prototype BbvI.

Screening of extreme environments in search for novel microorganisms may lead to the discovery of robust enzymes with either new substrate specificities or thermostable equivalents of those already found in mesophiles, better suited for biotechnology applications. Isolates from Iceland geysers’ biofilms, exposed to a broad range of temperatures, from ambient to close to water boiling point, were analysed for the presence of DNA-interacting proteins, including restriction endonucleases (REases). GeoICI, a member of atypical Type IIS REases, is the most thermostable isoschizomer of the prototype BbvI, recognizing/cleaving 5 -GCAGC(N8/12)-3 DNA sequences. As opposed to the unstable prototype, which cleaves DNA at 30°C, GeoICI is highly active at elevated temperatures, up to 73°C and over a very wide salt concentration range. Recognition/cleavage sites were determined by: (i) digestion of plasmid and bacteriophage lambda DNA (λ); (ii) cleavage of custom PCR substrates, (iii) run-off sequencing of GeoICI cleavage products and (iv) shotgun cloning and sequencing of λ DNA fragmented with GeoICI. Geobacillus sp. genomic DNA was PCR-screened for the presence of other specialized REases-MTases and as a result, another putative REase- MTase, GeoICII, related to the Thermus sp. family of bifunctional REases-methyltransferases (MTases) was detected.

Relationship between APE1 single nucleotide polymorphism and prognosis of primary small cell carcinoma of esophagus / 中国综合临床

Objective To examine the single nucleotide polymorphism(SNP) of apurinic/apyrimidinic endonuclease1 (APE1) in primary small cell carcinoma of esophagus(PSEC) ,then investigate the relationship between these SNPs and the prognosis.Methods Sixty cases first-treated patients with PSEC were recruited, patients with esophageal squamous cell carcinoma (ESCC) and healthy blood donors were recruited as positive and negative controls.APE1 (Asp148Glu) of the patients with PSEC and controls were genotyped by the TaqMan method.Every patient was treated with platinum-based chemotherapy(EP regimen for PSEC and TP regimen for ESCC)and radiotherapy(3D-CRT) ,then every case was followed-up for 2 years.The relationship between these SNPs and the follow-up outcome was analyzed.Results Compared with the ESCC group and control group, APE1 148 pure mutant(Glu/Glu) of PSEC group increased significantly(PSEC group was 40％ (12/30), ESCC group was 13.3％ (4/30) , control group was 10％ (2/20)), the difference was statistically significant (x2 =7.248,P =0.027).According to data of following-up, there was a significant increase in rate of progress (1year:40.0％ (12/30) vs 16.7％ (5/30), x2 =4.022, P =0.045;2 years: 86.7％ (26/30) vs 40.0％ (12/30) ,P =0.004) and a significant decrease in survival (33.3％ (10/30) vs 76.7％ (23/30)) of PSEC compared with ESCC.The SNPs of APE1 Asp148Glu was significantly correlated with frequency of progress, a significant increase was found in rate of progress of the patients with mutant type(Asp/Glu±Glu/Glu) compared with wild genotype(1 year: 50.0％(11/22) ,x2 =3.854,P=0.05;2 years: 81.8％ (19/22) ,x2 =10.519,P =0.001) ,the survival of the patients with mutant genotype was significantly lower than wild type (22.7％ (5/22) ,x2=10.77,P=0.001).Conclusion The most of polymorphisms of APE1(Asp148Glu) are mutation type in PSEC.Pure mutant genotype (APE1 148Glu/Glu) carry significant enhancement of progression.The polymorphisms of APE1 (Asp148Glu) maybe one of those molecular mechanisms of high frequency of progress and poor prognosis in PSEC.

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.

Expression and activity assay of recombinant flap endonuclease 1 / 生物工程学报

Flap endonuclease 1 (FEN1) is an endonuclease that catalyzes invasive reaction. It can be used in signal-amplification reaction-based nucleic acid assay. However, the application of FEN1 is hampered due to the lack of detailed protocols to express and purify the enzyme, and to quantify the enzyme activity. In this paper, the DNA fragment coding the gene of FEN1 from Archaeoglobus fulgidus was synthesized, and inserted into the plasmid of pET24a(+) to express recombinant FEN1 with His-tag. After optimizing the expression, detailed expression protocol of FEN1 was obtained by culturing the recombinant E. coli at 37 ℃ with 200 r/min of shaking for 8 h, followed by inducing with 0.05 mmol/L IPTG at 37 ℃ for 11 h. The purified recombinant FEN1 with the molecular mass of 38 kDa was obtained by Ni-affinity chromatography. Moreover, we developed a accurate quantification method with fluorescence-labelled probes. Finally, the recombinant FEN1 was used in real-time PCR coupled with high specific invader assay for aldh2 gene genotyping to obtain the correct typing results, indicating that the recombinant FEN1 can be used in gene polymorphism detection. We provide a reliable enzyme for developing invasive reaction-based nucleic acid assay.

Plasmid and Restriction Endonuclease Analyses of Bacteria Associated with Pneumonia in HIV/AIDS Patients in Ekiti State, Nigeria.

Bacterial infections are an important cause of morbidity and mortality in individuals with the human immunodeficiency virus. The emergence of multiple-drug resistant bacteria has been documented by many researches. This study was therefore carried out to determine whether the resistances of bacterial isolates from HIV positive and HIV negative patients are plasmid mediated or chromosomal mediated. The Plasmid, Post Plasmid-curing Sensitivity and Restriction enzymes endonuclease were done using standard methods. The result of plasmid analysis showed that Plasmid-mediated resistance was observed in both populations and the molecular weight of the plasmid DNA was 1000 base pairs. Plasmid mediated resistance was common, and this was observed in all isolates from HIV/AIDS patients with exceptions of P. aeruginosa in which the resistance was chromosomally mediated. Restriction endonuclease analysis from E. coli revealed 3 distinct clusters. The result of restriction enzymes analysis indicate that the pneumonia infection in HIV/AIDS patients is likely to be hospital acquired in the study location. The study also suggests a common source of infection of the patients.

APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that shows elevated expression in a number of cancers. We attempted to determine whether serum APE1/Ref-1 is elevated in patients with bladder cancer. MATERIALS AND METHODS: Serum APE1/Ref-1 levels were determined using enzyme-linked immunosorbent assay in serum from patients with bladder cancer who had not received chemotherapy or radiotherapy (n=51) and non-tumor controls (n=55). The area under the receiver operating characteristic area under the curve was applied to determine the correlation between clinical factors and the serum levels of APE1/Ref-1. RESULTS: Serum levels of APE1/Ref-1 in bladder cancer patients were significantly elevated compared to those of the control group (3.548+/-0.333 ng/100 muL [n=51] for bladder cancer vs. 1.547+/-0.319 ng/100 muL [n=55] for the control group), with a sensitivity and specificity of 93% and 59%, respectively. Serum APE1/Ref-1 levels are associated with tumor stage, grade, muscle invasion, and recurrence. CONCLUSION: Serum APE1/Ref-1 might be useful as a potential serologic biomarker for bladder cancer.