Novel Halophyte EREBP/AP2-type DNA Binding Protein Improves Salt Tolerance in Transgenic Tobacco

EREBP/AP2-type proteins are members of a large DNA binding protein (DBP) family found in plants. Some members like APETALA2 and AtDREB/CBF can regulate flower development and response to environmental stresses, respectively. To characterize transcription factors involved in plant responses to salt stress, we constructed cDNA library from salt-treated halophyte (Atriplex hortensis) and isolated a novel gene encoding EREBP/AP2-type protein from this library. This cDNA contained an ORF of 723 bp and a long 3'-Untranslated-Region (UTR) of 655 bp. The deduced amino acid sequence showed one conserved DNA binding domain of EREBP/AP2, thus the corresponding gene was named AhDREB1 with a calculated molecular mass of 26.1 kD. AhDREB1 under the control of CaMV 35S promoter was then transformed into tobacco and nine independent transgenic lines were obtained and subjected to long term salt stress. The results suggested that overexpression of AhDREB1 improved the salt tolerance in transgenic tobacco through functioning as a regulatory molecule in response to salt stress. Analysis of Arabidopsis genome in database resulted in dozens of EREBP/AP2-type homologous proteins, of which seven members showed high similarity to AhDREB1. Secondary structure analysis predicted similar arrangement of a-helix in their DNA binding domains.

Analyses of Protein and Oil of Maize Offsprings Plants Introducing Soybean DNA

[Objective] The experiment was aimed to explore character variation between different families after DNA introduction and select variant plants with good stability. [Method] The method of pollen-tube-pathway was used to introduce total DNA of soybean into normal maize inbred line 7313 for selecting generation by generation. When field characters of maize, grain colors, grain traits and panicle axis colors were stable, the crude protein, gliadin, glutelin and oil content of grains which were selected from variant strains were detected and compared. [Result] The grain crude protein, gliadin, glutelin and oil content of line 26h-4-3 were significantly different from these of control treatment. The increments of D3 and D4 generation were 10.34% and 26.70%, 6.58% and 6.28%, 15.09% and 70.34%, 55.82% and 51.52% respectively. All indexes of line 26h-3-1 were also higher than these of control treatment and the increments of D3 and D4 generation were 5.67% and 21.63%,1.91% and 2.31%, 10.85% and 62.27%,22.49% and 9.67%. [Conclusion] The crude protein, gliadin, glutelin and oil content of variant line 26h-4-3 and 26h-3-1 were stable, so variant line 26h-4-3 and 26h-3-1 were excellent variant strains which satisfied the requirement of high protein breeding.

Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

Taq DNA聚合酶作为实时荧光定量聚合酶链式反应(qPCR)技术的核心组分,其性能优劣直接影响qPCR技术的进一步发展。然而,野生型Taq DNA聚合酶的耐抑制剂性能差、延伸性能不足。为获得具有高性能的Taq DNA聚合酶,采用基因工程技术将双链DNA结合蛋白Sso7d或Sto7d融合在野生型Taq DNA聚合酶的N端或C端,构建了4个均可溶表达的改造体,再经过耐受性测试筛选较优的改造体,结果显示:改造体Taq-Sto的耐受性最高,其热稳定性不受影响,且在1 s/kbp的延伸条件下能成功扩增靶标,表明Taq-Sto具有增强的延伸性能,在TaqMan探针法qPCR体系中对腐殖酸、单宁酸、全血等抑制剂同样表现出良好的耐受性。EMSA实验发现:Taq-Sto对DNA模板的结合亲和力有所提高,有利于增强Taq-Sto对模板的竞争力;将Taq-Sto应用于非洲猪瘟病毒(ASFV)的TaqMan探针法qPCR检测,与商品化试剂相比,Taq-Sto具有更低的ASFV检出限,且在体积分数为2%~6%的猪粪便样本或猪肉样本中的检测灵敏度分别为100.0%和85.4%,说明Taq-Sto在直扩qPCR检测领域更具有优势。

Endonuclease-rolling circle amplification-based method for sensitive analysis of DNA-binding protein

A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA （ssDNA） were spotted on a microarray. The endonuclease recognition site （ERS） and the DNA-binding sites （DBS） were arranged side by side within the duplex region. The working principle of the detection system is described as follows： when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification （RCA）. When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.

Neuronal effects of SP600125 pretreatment in a rat model of cerebral ischemia/reperfusion injury Inhibited down-regulation of DNA repair protein

BACKGROUND： Recent studies have shown that the selective inhibitor of c-Jun N-terminal kinases （JNKs） signaling pathway, SP600125, exhibits neuronal protective effects in a rat model of brain ischemia/reperfusion. OBJECTIVE： To determine the mechanisms of neuroprotective effects of SP600125 in a rat model of brain ischemia/reperfusion, and determine the role of the JNK signaling pathway in SP600125-induced effects. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Animal Experiment Center, Medical School of Xi＇an Jiaotong University from June 2007 to September 2008. MATERIALS： SP600125 was provided by Biosource, USA; rabbit anti-phospho-JNK （Thr183/Tyr185） polyclonal antibody from Cell Signaling Technology, USA; rabbit anti-X-ray repair cross-complementing protein 1 （XRCC1） and anti-Ku70 polyclonal antibodies from Santa Cruz Biotechnology, USA; and TUNEL kit from Beijing Huamei Biology, China. METHODS： A total of 108 male, 4-month-old, Sprague Dawley rats were randomly assigned to three groups, with 36 rats per group. The sham operation group and ischemia/reperfusion group （I/R group） were intracerebroventricularly injected with 10 μL 1% DMSO. The SP600125-treated group （pre-SP group） was given 10 μL SP600125 （3 μg/μL）. Thirty minutes later, brain ischemia was induced in the I/R and pre-SP groups using the four-vessel occlusion method. Specifically, whole brain ischemia was induced for 6 minutes, and the clips were released to restore carotid artery blood flow. Rats from each group were observed at 2, 6, 12, 24, 48, and 72 hours, with 6 rats for each time point. The sham operation group was treated with the same surgical exposure procedures, with exception of occlusion of the carotid artery. MAIN OUTCOME MEASURES： Hematoxylin-eosin staining was used to observe neuronal survival in the hippocampal CA1 region, TUNEL was used to detect apoptosis in the hippocampal CA1 region, and immunohistochemistry was used to detect expression of phospho-JNK, XRCC1, and Ku70. RESULTS： Following brain ischemia/reperfusion, neuronal survival significantly decreased, and the number of apoptotic cells significantly increased （P 〈 0.01）. Compared with the I/R group, neuronal survival significantly increased in the pre-SP group, and the number of apoptotic cells significantly decreased （P 〈 0.01）. Expression of phospho-JNK increased, and XRCC1 and Ku70 significantly decreased （P 〈 0.05） following ischemia/reperfusion. Compared with the I/R group, expression of phospho-JNK decreased, and XRCC1 and Ku70 significantly increased in the pre-SP group （P 〈 0.05）. Correlation analysis revealed an inverse correlation between phospho-JNK gray value and XRCC1 and Ku70 gray values in the hippocampal CA1 region （r = -0.983, -0.953, P 〈 0.01）. CONCLUSION： SP600125 treatment decreased apoptosis induced by global brain ischemia/reperfusion in the rat hippocampal CA1 region. Results suggested that the neuroprotective effects were due to inhibited phosphorylation of JNK and reduced down-regulation of XRCC1 and Ku70.

Chromodomain-helicase-DNA binding protein 5, 7 and pronecrotic mixed lineage kinase domain-like protein serve as potential prognostic biomarkers in patients with resected pancreatic adenocarcinomas

Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.

Pse-in-One 2.0: An Improved Package of Web Servers for Generating Various Modes of Pseudo Components of DNA, RNA, and Protein Sequences

Pse-in-One 2.0 is a package of web-servers evolved from Pse-in-One (Liu, B., Liu, F., Wang, X., Chen, J. Fang, L. & Chou, K.C. Nucleic Acids Research, 2015, 43:W65-W71). In order to make it more flexible and comprehensive as suggested by many users, the updated package has incorporated 23 new pseudo component modes as well as a series of new feature analysis approaches. It is available at http://bioinformatics.hitsz.edu.cn/Pse-in-One2.0/. Moreover, to maximize the convenience of users, provided is also the stand-alone version called “Pse-in-One-Analysis”, by which users can significantly speed up the analysis of massive sequences.

Inhibition of DNA-dependent Protein Kinase Catalytic Subunit by Small Molecule Inhibitor NU7026 Sensitizes Human Leukemic K562 Cells to Benzene Metabolite-induced Apoptosis

Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.

EFFECT OF ACTIVE COMPOUNDS ISOLATED FROM PTERIS SEMIPINNATA L ON DNA TOPOISOMERASES AND TYROSINE PROTEIN KINASE AND EXPRESSION OF C-MYC IN LUNG ADENOCARCINOMA CELLS

Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

A novel detection of single-stranded DNA binding protein based on ss-DNA modified chip using surface plasmon resonance microscopy

An ss-DNA gold chip was prepared based on self-assembly of the thiol-derivatized oligonucleotide, and used for the determination of single-stranded binding protein （SSB） by surface plasmon resonance microscopy （SPR）. The experiment results showed that SSB binds ss-DNA with high specificity, and relative signal of SPR response is proportional to the concentration of SSB in the range of 0.1-100 ng/mL with a detection limit （S/N = 3） of 0.07 ng/mL.

Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

REGULATED PHOSPHORYLATION OF THE GATA－2 DNA BINDINGPROTEIN IN ENDOTHELIAL CELLS

Endothelin-1 and a number of other genes expressd primarily in endothelial cells(EC)require a functional GATA element in their promoter region.The widely expressed zinc finger DNA binding protein GATA-2 has been characterized as the likely GATA factor which binds these GATA elements.To understand the specificity of this interaction,and to investigate the potential for regulation of GATA-2 activity,we have studied translation and post-translational modification of the GATA-2 protein. A specific antiserum immunoprecipitated a 52kDa GATA-2 protein from [35-S] methionine-labeled EC,as well as a wide variety of cultured human cell lines which express GATA-2 mRNA. Immunoprecipitation experiments with [32-P]-orthophosphate labeled cells indicated that GATA-2 is similarly phosphorylated in EC and non-EC lines. Thus the apparent cell-specific activity of this transcription factor is not regulated by translation or phosphorylation, and must derive from the interaction of GATA-2 with other nuclear proteins in the EC.Further studies investigated the potential regulation of GATA-2 phosphorylation in EC. Phosphoamino acid analysis indicated that GATA-2 is phosphorylated on serine and threonine residues in EC.The hasal phosphorylation of GATA-2 was rapidly and markedly increased when EC were treated with calcium ionophore A23187, while phorbol ester and forskolin had no effect.Phosphopeptide map analysis showed that A23187 induced phosphorylation of at least two additional sites in GATA-2.Gel shift assays employing nuclear extracts isolated from EC that had been treated with A23187 had a different DNA binding pattern when compared to control.This regulated phosphorylation of GATA-2 may provide a signaling pathway for hormonal regulation of endothelial cell genes such as endothelin-1 which alter their rate of transcription in response to increased intracellular calcium.

Studies on DNA Vaccine of the Conservative Region of Gene Encoding the Male-Specific Gynecophoral Canal Protein of Schistosoma japonicum (Chinese strain)

In order to observe the protective effect induced by vaccinating animals with the DNA vaccine of Sex-specific expression gene of Schistosoma, A 868 bp cDNA fragment amplified by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNA was cloned into the eukaryotic expression vector pcDNA3 and the recombinant eukaryotic expression vector pcDNA3-SjGCP1 was directly injected into BALB/c mice intramuscularly 3 times with the interval of 3 weeks .Both the vaccinated and control group of mice were challenged with 40 cercariae of Sj 5 weeks after last injection and perfused 7 weeks post-challenge. The worm and egg reduction rate got from vaccinated mice was 32.4% and 46.9% respectively. The result indicated that pcDNA3-SjGCP1 DNA vaccine induces the significant protection in animal against Schistosoma japonicum infection.

Potential role of DNA-dependent protein kinase in cellular resistance to ionizing radiation

In this paper,we study the ability of DNA-PK-deficient(M059J) and -proficient(M059K) cells to undergo the rate of cellular proliferation,cell cycle distribution and apoptosis after 10 Gy X-ray irradiation,and the role of DNA-PK in radiosensitivity.The results showed that M059J cells exhibited hyper-radiosensitivity compared with M059K cells.A strong G2 phase arrest was observed in M059J cells post irradiation.Significant accumulation in the G2 phase in M059J cells was accompanied by apoptosis at 12 h.Altogether,the data suggested that DNA-PK may have two roles in mammalian cells after DNA damage,a role in DNA DSB repair and a second role in DNA-damaged cells to traverse a G2 checkpoint,by which DNA-PK may affect cellular sensitivity to ionizing radiation.

Effect of Manganese on DNA-Protein Crosslinks of Testicle in Chicken

In order to explore the effect of manganese on DNA-protein crosslinks （DPC） of testicle in chicken. 500, 800, 1 700 mg·kg^-1 MnC12 were added to forage to establish the model of the sub-chronic manganese poisoning. After 30, 60 and 90 d, testicles were taken out to detect hydroxyl radical inhibiting capacity and DPC content. The results showed that compared with control group, hydroxyl radical inhibiting capacity significantly decreased and DPC content notably increased, and there was a time-dose relationship. It demonstrated that manganese could decrease the inhibitng capacity of hydroxyl radical, increase the content of hydroxyl and DPC, and induce DNA damage.

Study on Binding Affinity of a Glutathione <i>S</i>-Transferase (GST) Fusion Protein to DNA Probe

The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli;ii) quantify the protein by SDS PAGE and Bradford assay;iii) determine protein-DNA interaction of the purified protein by Electrophoretic Mobility Shift Assay. Bacterial culture prepared by inoculation of a single E. coli colony that had a GST fusion protein (gst: six-X10 hd) constructed by ligation of the six-7-hd (X10) sequence into the BamHI and EcoRI sites of the vector pGEX-2T, grown overnight, was sonicated using Cole-Palmer Ultrasonic Homogenizer. Fusion protein was eluted from the beads with Tris-glutathione buffer (50 mM Tris [pH 8.1], 20 mM glutathione), which contained reduced Glutathione. SDS-PAGE was used to calculate the extracted bound protein. Total protein quantification was then estimated by the Bradford assay. Bovine Serum Albumin (BSA) absorbance values were used to plot the standard curve used to calculate the concentrations of the sample proteins. Nylon membrane was used for the electrophoretic transfer;membrane was cross linked and detected by Pierce’s Chemiluminescent Nucleic Acid Detection module. Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples;however, there was no shift in the bands for the wild-type, positive control. The concentration of the elute 1 was obtained by the Bradford assay as 242.52 ng/μl and that of elute 2 was 106.30 ng/μl. Similarly, the result obtained by gel analysis was 300 ng/μl (0.3 μg/μl) and 150 ng/μl (0.15 μg/μl) for elutes 1 and 2 respectively.

Construction and in vitro Expression of Streptococcus Mutans Surface Protein Encoding DNA Vaccine

DNA vaccine plasmids were constructed that encoded two highly conservative regions of a surface protein, PAc, from the human major cariogenic bacterium, Streptococcus mutans . Antigen expression was evaluated in vitro by immunohistochemical analysis of human endothelial cells following cationic liposome mediated transient transfection with recombinant plasmid. The results of this study provided a basis for further testing of these recombinant plasmids in primates and for efficacy testing of dental caries DNA vaccines in human volunteers in future.

Influence of edaravone on growth arrest and DNA damage-inducible protein 34 expression following focal cerebral ischemia-reperfusion in rats

Objective:To investigate the influence of edaravone on the expression of growth arrest and DNA damage-inducible protein 34(GADD34).Methods:A total of 108 healthy male Sprague-Dawlcy rats were randomly divided into sham operation group,model group and edaravone.group(36 cases for each group).Transient focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 h followed by reperfusion in Sprague-Dawlev rats.Then.GAOD34 expression was measured with immunohistochemistry at different time-points after reperfusion in the peri-infarct regions of all rats.Results:The GADD34 expression was detected in the peri-infaret regions of rats 1 h after reperfusion,which reached its peak 24 h after reperfusion.And edaravone could significantly down-regulate the GAOD34 expression.Conclusions:Edaravon could down-regulate GADD34 expression,which suggests that edaravone may exert an important function in inhibiting endoplasmic reticulum stress reaction by scavenging free radicals in the upper stream.

七种蛋白酶抑制剂对多房棘球蚴DNA损伤诱导样1蛋白活性的影响

目前,多房棘球蚴病(alveolar echinococcosis, AE)尚无有效药物治疗手段,迫切需要开发新型治疗药物。前期研究表明,HIV蛋白酶抑制剂(HIV protease inhibitors, HIV PIs)具有潜在抗寄生虫功能。本文旨在研究HIV PIs对Echinococcus multilocularis(Emu)DNA损伤诱导样1蛋白(DNA damage inducible 1 protein, Ddi1)活性的影响。本研究通过构建真核表达重组载体pFastBac1-Emu Ddi1,在昆虫细胞系Sf9细胞中表达筛选出P1代和P2代,纯化出可溶性Ddi1重组蛋白,然后与目的蛋白的荧光底物检测纯化蛋白的活性,进一步检测沙奎那韦(saquinavir, SQV)、利托那韦(ritonavir, RTV)、安普那韦(amprenavir, APV)、阿扎那韦(atazanavir, ATV)、洛匹那韦(lopinavir, LPV)、福沙那韦(fosamprenavir, Fos)、达芦那韦(darunavir, DRV)等7种HIV PIs对Emu Ddi1重组蛋白活性的抑制能力。结果显示:细胞系内真核表达产物的酶活Km为1.422μmol·L^(-1),具有良好的亲和力和活性,最终筛到沙奎那韦对Ddi1蛋白二聚体活性位点的抑制率达67%,其IC_(50)为34,说明沙奎那韦对Emu Ddi1重组蛋白酶活性具有良好的抑制效果。以上结果提示:沙奎那韦抑制重组蛋白Ddi1的活性,可能成为Ddi1的靶向药物,以期为替代药物或开发联合用药提供基础。

Excess adsorption of biomolecules on soft surfaces: Adsorption of DNA, proteins and lactose on fatty surfaces

Insoluble fatty surfaces are involved in many important interactions such as in biomembranes with soluble biological macro and micromolecules. In this paper we have studied the adsorption interaction of aqueous solution of DNA, some proteins and lactose on several sparingly soluble fatty substances namely milk fat, stearic acid, palmitic acid, phosphatidyl choline and cholesterol surfaces by measuring the depletion of the adsorbates by analytical methods. Adsorption () of DNA on the soft surfaces of stearic acid, milk fat, phosphatidyl choline, palmitic acid and cholesterol was measured as a function of DNA concentration C2. In each case was found to increase with C2 until it reached the maximum value at a critical concentration . For different surfaces stands in the order: stearic acid > milk fat > phosphatidyl choline > cholesterol > palmitic acid. DNA forms multilayers on stearic acid surface. Adsorption of hemoglobin on cholesterol surface is found to be negative or zero but that of BSA on cholesterol is positive. Adsorption of gelatin on cholesterol surface is significantly higher than that of BSA. Lysozyme on cholesterol surface forms multilayers and on casein forms bilayer. The lowering of free energies ?DGo for all systems have been calculated using integrated form of the Gibbs adsorption and their values have been compared with each other. It is concluded that despite differences in the adsorption behavior of the biomolecules on various soft surfaces, free energy change expressed as Bull’s free energy change (Δ) remain nearly constant except for BSA-fatty acid interaction which may be likely due a specific interaction.