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Extrachromosomal DNA (ecDNA) is a small segment of circular DNA located outside the chromosome, which has the function of self-replication. Recently, amplification of oncogenes on ecDNA has been proved to be a common phenomenon in tumor cells, and has some characteristics worth studying, such as correlation with patients' poor prognosis. Multiple chromosomal events are involved in the formation of ecDNA, and its amplification can directly increase the number of DNA copies of extra-chromosomal oncogenes and accelerate the generation and development of tumors. Moreover, the segregation pattern of unequal transmission of parental ecDNA cells to offspring not only increases tumor heterogeneity, but also enhances tumor adaptation to environment and response to therapy. This article reviews the current status and potential significance of ecDNAin tumor cells. [ABSTRACT FROM AUTHOR]

DNA sensor, a kind of pattern recognition receptor (PRR), is widely expressed in innate immune cells. It activates the inflammatory signaling pathways and triggers an innate immune response by recognizing the pathogens or DNA in abnormal host cells. DNA-dependent activator of IFN-regulatory factors (DAI) is the first cytoplasmic DNA receptor discovered, which plays an important role in regulating the innate immune responses characterized by induction of interferon and programmed cell death. The article summarizes the molecular characteristics of DAI, its downstream signaling pathways, and its role and mechanism in anti-infective immunity, tumor immunity and inflammatory diseases. It also makes a preliminary exploration of the correlation between DAI and transplantation immunology, and provides a new target for the therapy of various immune diseases.

In order to examine the toxic effects of p-xylene on the hepatopancreas of Haliotis discus hannai , a subchronic toxicity test was carried out for 21 days by setting 4 concentrations (0.5, 1.0, 1.5, and 2.0 mg·L -1 ) and the control group. DNA damage in hepatopancreas cells of H. discus hannai was analyzed by comet assay. The CASP analysis software was used to analyze the damage indices, including tail rate, tail length, percentage of tail DNA, and olive moment. The results showed that DNA damage in hepatopancreas cells occured in all exposed groups, with different degrees of damage. The DNA damage of hepatopancreas cells was aggravated with the increase of exposure concentration. High concentration of p-xylene could induce apoptosis, showing a certain dose-damage effect. Short-term exposure to medium concentration of p-xylene could cause DNA damage in hepatopancreas cells of H. discus hannai , and the damage degree increased with the extension of exposure time, showing a certain time-damage effect. However, the damage indices of DNA decreased after prolonged exposure, which might be related to the DNA repair mechanism of the cell and the metabolic mechanisms of the detoxification system. In conclusion, p-xylene could induce oxidative damage in hepatopancreas cells of H. discus hannai , resulting in DNA fragmentation. Prolonged exposure to high concentration of p-xylene could lead to apoptosis.

Without losing its high resolution, high-speed atomic force microscope (HS-AFM) represents a perfect combinationof scanning speed and precision and allows real-time and in situ observation of the dynamic processes in a biological system atboth the cellular and molecular levels. By combining the extremely high temporal resolution with the spatial resolution andcoupling with other advanced technologies, HS-AFM shows promising prospects for applications in life sciences such as cellbiology. In this review, we summarize the latest progress of HS-AFM in the field of cell biology, and discuss the impact ofenvironmental factors on conformation dynamics of DNA, the binding processes between DNA and protein, the domainchanges of membrane proteins, motility of myosin, and surface structure changes of living cells.

Objective: To express and purify FOXO1 DNA binding domain (DBD) and to evaluate its DNA binding properties.
 Methods: Gene sequence of FOXO1-DBD was optimized and the recombinant strains were induced at low temperature to obtain soluble protein of FOXO1-DBD. Then, FOXO1-DBD protein was purified through Ni affinity chromatography and cation exchange. Finally, the DNA binding properties of FOXO1-DBD was evaluated by electrophoretic mobility shift assay (EMSA).
 Results: Most of soluble proteins were obtained by optimizing its genes sequence and induction at 21 ℃. Over 95% purity of FOXO1-DBD protein was obtained through two-steps purification. Purified protein exhibited good binding property to the DNA binding motif (G/ATAAACA) of FOX family.
 Conclusion: An efficient expression and purification method for FOXO1-DBD is established, and the complexity of FOXO1 protein in recognition of DNA is verified.

The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, "Helper", to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.

Objective: To study the structural characteristics of the mouse DNA-dependent activator of interferon-regulatory factors (DAI) and its related molecular mechanism in anti-viral innate immune responses and signal transduction., Methods: The coding sequence of mouse DAI gene was amplified from splenic mononuclear cells by reverse transcription-PCR, and the genetic evolution and molecular structure of the mouse DAI gene were analyzed by bioinformatics softwares. After mouse DAI was stimulated by poly(dA-dT) and poly(dG-dC), the nuclear factor κB (NF-κB) and interferon-beta (IFN-β) promoter-driven luciferase activity were detected by dual-luciferase reporter assay system., Results: The open reading frame (ORF) of the cloned mouse DAI sequence was 1236 bp, encoding 411 amino acids, which exhibited identity with the corresponding sequences of cattle, pig, rat and other mammals ranging from 60%, 63.1%, 84%, and it contained two Z-DNA domains (Zα and Zβ), DNA binding region (D3) and signaling domain (SD). The stimulation of poly (dA-dT) increased the expressions of mouse DAI activated transcription factors NF-κB and IFN-β promoter. However, the stimulation of poly(dG-dC) only induced the activation of NF-κB but not IFN-β promoter., Conclusion: Mouse DAI as an important cytosolic DNA sensor, is responsible for the recognition of A/T or G/C-rich DNA derived from DNA virus. It may play an important role in anti-viral innate immune responses.

Objective: To evaluate a novel method for in vitro generation and degradation of neutrophil extracellular traps (NETs), which are a newly recognized structure that is involved in the pathogenesis of autoimmune diseases and thrombosis., Methods: Neutrophils from peripheral blood of healthy donors were obtained by Ficoll-Histopaque gradient separation. NET release was initiated by phorbol myristate acetate (PMA) and validated by immunofluorescence staining and agarose gel electrophoresis. NETs degraded by DNase I and healthy human plasma were quantified by fluorescence spectrometry after staining with PicoGreen., Results: HE staining showed that the purity of neutrophils was up to 95% after Ficoll-Histopaque gradient separation. NET immunofluorescent staining revealed that the network structure was mainly composed of DNA and histones, with molecular length more than 10 kb as demonstrated by agarose gel electrophoresis. Moreover, both DNase and healthy human plasma could induce the degradation of NETs, in varying degrees., Conclusion: This work established an efficient method for in vitro generation and degradation of human NETs.

Objective: To clone and express a high mobility group box 1(HMGB1) protein of Schistosomajaponicum (Mainland strain) and analyze its function., Methods: The DNA fragment of open reading frame encoding Sj HMGB 1 protein was amplified by RT-PCR from the mRNA of S. japonicum worms, then it was subcloned into the expression vector pET28a(+) to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3), and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombinant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding capacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 protein and infected with 45 +/- 2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection, the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue, respectively. The worm and egg reduction rates were calculated respectively., Results: A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR, which was the open reading frame (ORF) encoding SjHMGBlprotein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA, and the recombinant protein immunized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abundantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effective immune protection against S. japonicum., Conclusion: The gene encoding HMGB1 from S. japonicum and the soluble recombinant SjHMGB1 protein with natural functional activity are obtained, and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

The DNA methylation patterns and levels are depended on the function of DNA methyltransferase and DNA demethylase, and DNA demethylase plays a critical role in active DNA demethylation. In this paper, all homologous DNA demethylase gene copies were identified in monocots (O. sativa and S. bicolor) and dicotyledon (A.thaliana and P. trichocarpa) based on ten known DNA demethylase genes from rice and Arabidopsis. Two types of new DNA demethy-lase-like genes (DML4 and DML5) were identified. Tandem duplication, segmental duplication and whole genome duplica-tion exist in DNA demethylase gene family in plants, which result in neofunctionalization and subfunctionalization upon the phylogeny of conserved glycosylase domains and chromosomal locations of genes. Furthermore, the expression of DNA demethylase genes was investigated in different tissues. This study will facilitate our understanding of the relationship be-tween function and evolution of DNA demethylase, and utilizing the DNA demethylase genes in plants.

Objective: To assess the diagnostic value of sperm DNA fragmentation (SDF) for male infertility., Methods: Two hundred and ninety-nine males attending infertility clinic were classified into 157 primary infertile cases and 142 fertile controls. Semen analysis was performed as recommended by the World Health Organization (WHO). SDF was assessed by sperm chromatin dispersion (SCD) assay, and the results were expressed as DNA fragmentation index (DFI)., Results: The DFI was significantly higher in infertile males than that in fertile controls [(17.1± 9.3)% vs. (14.2± 9.0)%](P< 0.01). No significant difference was detected in the age of male and female partners, seminal volume, sperm count, motility and morphology between infertile males and fertile controls (P> 0.05). The area under the receiver operating characteristic curve (AUC) was 0.861 [95% confidence interval (CI)= 0.814-0.907] for 15.1% of SDF. The threshold level of 15.1% was derived as cut-off value to discriminate infertile men from fertile controls. By this threshold, specificity was 88.2% and sensitivity was 81.8%. The 299 men were divided into group A (n= 120) with DFI≥ 15.1% and group B (n= 179) with DFI< 15.1% based on the cut-off value. The percentage of infertile men in group A was significantly higher than that in group B (79.2% vs. 34.6%) (P< 0.01). The odds ratio (OR) for infertility in the two groups was 7.2 (95%CI= 4.2-12.3)., Conclusion: Sperms with high-level of DNA fragmentation can impair male fertility. DFI can be used as a good diagnostic marker for male infertility.

Objective: DNA phosphorothioate modification means substituting a non-bridging oxygen with a sulfur in DNA. The modification endows DNA with such chemical property that protects the hosting bacteria against peroxide. The modification is controlled by a dnd gene cluster. Salmonella entericaserovar Cerro 87 is one of the bacteria that harbor the DNA phosphorothioate modification. The modification is carried out by dptB, C, DandE. Ourstudy is designed to clone and express dptC, to optimize the expressing condition, and then to purify the DptC., Methods: dptC DNA fragment was amplified by KOD PCR with the special primers and S. entericaserovar Cerro 87 genomic DNA template. A fusion expression vector pJTU3622 was constructed by inserting the dptC DNA fragment into pGEX-6P-1 inSmaI and XhoI sites. The positive clone was verified by antibiotics resistance gene screening and sequenced, and then transferred into host strain E. coli BL21 (DE3) pLysS to producean engineering bacterium Anxh103. After optimizing the expression condition for dptC, we purified DptC from Anxh103 by Aikta FPLC with a GST-Trap column., Results: A fusion expression vector pJTU3622 and an engineering bacterium Anxh103 were produced. The optimizing expressing condition for dptC is as follows: induced at 18 degrees C for 8 - 18 h; 0.6 mmol/L IPTG, LB with 50 micromol/L Fe2+., Conclusion: The anchor redeemed for high throughput expression of dptC. The TEV site in pJTU3622 made the process of purifying DptC easier and simpler. This helps lay the ground work for future study on the function of DptC. Also, the light brown color of DptC and the medium with 50 micromol/L Fe2+ showed us DptC has the same character with DndC which belongs to an iron-sufur protein with 4Fe - 4S.

In this study, we expressed and purified supercharged green fluorescent protein (+36GFP) that we used to study its combination with nucleic acid and its cell transduction efficiency as carrier of DNA. We transformed pET+36GFP-HA2 plasmid into Escherichia coli BL21 (DE3), then expressed and purified the target protein. We used the protein to transduce a variety of mammalian cell lines including B16 cells, 293 cells, A549 cells and HepG2 cells at specified protein concentrations. Transduction efficiency of the protein was analyzed by flow cytometry. Under laser scanning confocal microscope, we observed visually transduction efficiency of +36GFP protein (100 nmol/L) to A549 cells. We incubated +36GFP with plasmid DNA and analyzed their binding ability with gel mobility shift assay. Then we transduced cells with the mixture of plasmid DNA/+36GFP protein at various ratio and detected the expression of reporter gene by using laser scanning confocal microscope and flow cytometry. The experimental results demonstrate that +36GFP had high transduction efficiency, and as the concentration increased, the efficiency improved in a dose-dependent manner. Gel mobility shift assay indicates that +36GFP could bind to plasmid DNA, blocking the migration of DNA in the gel in a concentration-dependent manner. After the plasmid wrapped by +36GFP penetrated into cells, the cells could express target protein efficiently, proving that +36GFP had the ability to carry nucleic acids into cells. Sucussful expression and purification of +36GFP protein confirms its high efficiency of cell transduction and its ability as carrier to deliver exogenous nucleic acids into cells.

Double-stranded DNA (dsDNA) microarray, also known as protein binding microarray (PBM), is an important technique that can be used to assay the interaction of DNA-binding protein (such as transcription factor, TF) with vast amount of DNA molecules in high-throughput format. This technique immobilizes large amount of various dsDNA molecules on the surface of a solid support (such as glass slide) for detecting the binding interaction of a DNA-binding protein with all of the immobilized dsDNA molecules, and thus determining the DNA-binding affinity, specificity and preference of TFs. In recent years, this technique has demonstrated its valuable applications in several aspects, including rapidly characterizing DNA-binding specificity of large number of TFs, building DNA-binding profiles of TFs, identifying DNA-binding sites and target genes of TFs, discriminating the subtle DNA-binding preferences of members and their dimmers of a TF family, and examining the effects of a cofactor on the DNA-binding specificity of TFs. This paper reviews the principal, techniques, and applications of dsDNA microarray.

The differential gene expression is the molecular base of development and responses to stimuli of organisms. Transcription factors (TFs) play important regulatory roles in this kind of differential gene expression. Therefore, to elucidate how these TFs regulate the complex differential gene expression, it is necessary to identify all target genes of them and construct the gene transcription regulatory network controlled by them. DNA binding is a key step for TFs regulating gene transcription. Therefore, in order to identify their target genes, it is indispensable to identify all possible DNA sequences that can be recognized and bound by TFs at the molecular level of their interactions with DNA, i.e., construction of the DNA-binding profiles of TFs. In recent years, along with the development of DNA microarray and high-throughput DNA sequencing techniques, there appeared some revolutionary new techniques for constructing DNA-binding profiles of TFs, which greatly promotes studies in this field. These techniques include ChIP-chip and ChIP-Seq for constructing in vivo DNA-binding profiles of TFs, dsDNA microarray, SELEX-SAGE, Bind-n-Seq, MMP-SELEX, EMSA-Seq, and HiTS-FLIP for constructing in vitro DNA-binding profiles of TFs. This paper reviewed these techniques.

Accurate estimation of the postmortem interval (PMI) has been one of the most important and complicated issues in the forensic practice. In order to provide novel perspectives for the future research concerning PMI, the advantages and disadvantages of related traditional methods, postmortem degradation of nucleic acid and tissue, the componential change of vitreous humor and histological biochemistry since 2002 have been introduced and compared in this review.

Objective: To develop a method for enriching methylated DNA in clinical samples using mesocellular silica foams (MCFs) immobilized with methyl-CpG binding domain (MBD)., Methods: MCFs with ultra-large pore size were synthesized, functionalized and immobilized with GST-MBD., Results: The large cage-like pore structures of MCF materials was retained after functionalization and immobilization, with pore diameter of 55 nm, window size of 30 nm, and a high pore volume of 1.0 cm(3)/g. The loading amount of MBD was as high as 53 wt%. Immobilized MBD showed high binding activity and stability. In a binding buffer with salt concentrations ranging 500-550 mmol/L, the MCF-MBD can selectively enrich methylated DNA from the mixed DNA solution., Conclusion: The MCF-MBD method may offer a better choice for high-throughout DNA methylation screening, and has laid a foundation for clinical application, prenatal diagnosis and research on DNA methylation-related genetic diseases.

A series of multinuclear diethylenetriamine ligands were synthesized and used as artificial nuclease enzyme model. Target compounds were characterized by 1H NMR, 13C NMR, IR and ESI-MS. Preliminary studies on the cleavage of pUC19 DNA in the presence of metal complexes have also been performed and the results revealed that these complexes could act as powerful catalysts for the cleavage of pUC19 DNA after 48 h under physiological conditions. The hydrolytic cleavage mechanism of DNA plasmid by title compound was confirmed by T4 DNA ligase experiment.

Objective: To explore the mechanism of cell death in Microcystis aeruginosa PCC 7806., Method: According to the water environment of the later period of algal bloom, M. aeruginosa PCC 7806 was treat with dark and O2 limitation. We observed the morphological changes using transmission electron microscope (TEM) and detected the reactive oxygen species (ROS) activity and Caspase3 activity in M. aeruginosa PCC7806 subjected to dark and O2 limitation. DNA status was also examined with the methods of Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and agarose gel electrophoresis., Result: Massive algae cell died after 48 h treatment under dark and O2 limitation. During cell death process, we observed some changes of cell organelles including ribosomes and thylakoids disorganization, cytoplasmic vacuolation, nucleoplasm diffusion and plasmolysis in M. aeruginosa PCC7806 subjected to darkness and O2 limitation. Meanwhile, we found that increased ROS reactivity and capase 3 activity were related to the cell death process of M. aeruginosa. DNA breakage and fragmentation were proved by TUNEL staining and agarose gel electrophoresis during cell death process., Conclusion: All results showed that cell death with characteristics similar to eukaryotic programmed cell death could be induced in M. aeruginosa PCC 7806 after treatment with darkness and O2 limitation. Therefore, we suggested that the mechanism of cell death are conserved during evolution according to the characteristics of cell death shared between eukaryotes and Microcystis.

Objective: The aim is to explore the self-regulation mechanism of the rex in Streptomyces rimosus M4018., Methods: We cloned the rex of S. rimosus M4018 (Sr-rex) based on its homologoussequence in Streptomyces coelicolor A3(2) and its upstream rex operator (ROP) fragment using PCR and genome walking. An electrophoretic mobility shift assay (EMSA) was applied to analyze the regulation of rex to ROP in vitro., Results: Sr-rex is 846 bp in length and has a 84% identity with the one in S. coelicolor A3(2) in amino acid sequence. It was deposited in Genbank under the accession number GQ849479. The expressed Sr-Rex by E. coli was mainly composed of alpha-helixes and beta-sheets, which was in compliance with the prediction. An Electrophoretic Mobility Shift Assay (EMSA) confirmed the specific binding activity of Sr-Rex with ROP. Meanwhile, we synthesized a 22 bp DNA fragment (ROP1) based on the minimal binding site of ROP. The maximal binding ratio of this fragment to Sr-Rex was 5:1 (molar). NADH negatively affected the binding activity, however, NAD+ had no impact on it., Conclusion: In S. rimosus M4018, the Rex regulated the gene expression of ROP via sensing the intracellular level of NAD (H).

Naturally transformable bacteria are able to take up DNA to acquire new genetic traits in the environment. To be naturally transformed, bacteria need to establish a physiological state, called natural competence, in which DNA uptake and processing genes are expressed. DNA uptake proteins assemble a complex to pull exogenous DNA into the cytoplasm where it can recombine with the genome DNA or establish as a plasmid. In general, DNA uptake of bacteria could be divided into two stages: DNA is transported from the milieu to the periplasm at the first stage (for Gram-negative bacteria) and is translocated across the inner membrane at the second stage. Our work and other studies revealed new plasmid DNA transformation modes in Escherichia coli. Here, we first reviewed recent advances in the molecular mechanism of natural transformation and then described the distinctive plasmid transformation mode in E. coli.

Accurate estimation of postmortem interval (PMI) is one of the most important and difficult issues in forensic medicine. After death, the tissues autolyze and biomacromolecules degrade. DNA concentration decreases gradually with linear relationship with PMI. The housekeeping gene mRNA, for example beta-actin, GAPDH, has certain stability and can be used to PMI estimation as internal standard. This paper reviews the research progress and problems about DNA, RNA and proteins in the estimation of PMI in order to provide guidance for forensic pathology.

Circular dichroism (CD) is an useful technique for monitoring DNA conformation changes resulting from changes in environmental conditions, such as temperature, ionic strength, and pH, and also for the study of the interaction between DNA and ligands (including small molecules and proteins). CD spectroscopy of DNA arises from the asymmetric backbone sugars and by the helical structures often adopted by nucleic acids. By the interpretation of induced circular dichroism (ICD) of ligand signals resulting from the coupling of electric transition moments of the ligand, DNA bases within the asymmetric DNA environment, ligand-DNA interactions, as well as the DNA-binding mode can be assessed. A number of important conclusions have been reported that related to the observed ICD signals resulting from the interactions between intercalators and groove binders with DNA. If short oligonucleotide sequences are used in the study, sequences-specific of binding also can be deduced. CD determination requires smaller amounts of sample, and not limited by the molecular weight or size and can be performed rapidly; though CD is of low resolution, but it's a complement to NMR and X-ray diffraction methods. This review will introduce the characters of the CD spectra of DNA, and its application to the studies of DNA with small molecules; some progress of the studies in our laboratory will also be discussed. CD is expected to be used as a screening method in seeking more DNA-targeted drugs, such as, antineoplastic, antimicrobial and antiviral drugs.

An ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the analysis of biomarker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) in deoxyribonucleic acid (DNA) was developed. The artificial oxidation of 2'-deoxyguanosine (dG) at numerous stages in the sample preparation bring a challenge to the accurate measurement of 8-OHdG in DNA. To avoid the artificial oxidation during the enzymatic digestion, desferrioxamine mesylate as a protectant was added into the mixtures. By utilizing YM-3 Centricon membrane (3 000 of relative molecular mass cut off), excess proteins in enzymatic solutions were effectively removed, allowing direct UPLC-MS/MS analysis. The UPLCMS/MS analysis showed a linear relationship between the peak areas and the amounts of 8-OHdG in the range of 17.6-1400 fmol, and the correlation coefficient was 0.9998. By using the developed method, the content of 8-OHdG in calf thymus DNA (CT DNA) was estimated about 12.9 +/- 2.35 (calculated as 8-OHdG/10(6) dG), which was consistent with the previous work. This method can also be applicable for the detection of 8-OHdG in DNA under various oxidative stresses.

The control of gene transcription is a critical level of gene expression regulation. The interactions between transcription factors (TF) and their DNA binding sites (TFBS) play a key role at this level. In order to decipher the molecular mechanism of the interactions of TFs with TFBSs and construct transcription regulatory network, it is necessary to systematically collect, save, and analyze the information of discovered TFs and their TFBSs. In recent years, multiple TF and TFBS-related databases have been established. These databeses significantly promoted the TF-related studies in the fields of molecular biology, bioinformatics, and system biology. This paper summarized the contents, characteristics, access, and advances of main TFs and TFBSs-related databases, including TRANSFAC, JASPAR, TFDB, TRRD, TRED, PAZAR, MAPPER and others.

Objective: To investigate the role of nuclear factor-ΚB (NF-ΚB) in severe pneumonia and observe the effects of Xuebijing injection in its treatment., Methods: Thirty hospitalized patients with severe pneumonia were divided into the routine therapy group (n=14) and Xuebijing therapy group (n=16) in whom with Xuebijing injection 100 ml was given once daily for 7 days besides routine therapies, according to the random numeral. The DNA binding activity of NF-ΚB in human monocytes was detected before and 3 days and 7 days after administration, the contents of tumor necrosis factor-α (TNF-α), procalcitonin (PCT) and C-reactive protein (CRP) were determined, and the changes in coagulatory and fibrinolytic parameters were assayed at the same time. Acute physiology and chronic health evaluationII (APACHEII) score was also recorded. Ten healthy volunteers served as the healthy control group., Results: The DNA binding activities of NF-ΚB, the contents of TNF-α, PCT, CRP, fibrinogen (Fib), D-dimer in hospitalized subjects with severe pneumonia were higher before treatment than those in healthy control group, while the prothrombin time (PT), thrombin time (TT) were significantly lower (P<0.05 or P<0.01). Compared with the routine therapy group, the DNA binding activity of NF-ΚB (grey level) at the 7 days (66.60±36.23 vs. 79.90±39.11) was notably decreased in Xuebijing therapy group; the levels of TNF-α (ng/L, 25.81±11.67 vs. 33.78±13.36), PCT (μg/L, 1.91±1.09 vs. 2.96±1.80), CRP (mg/L, 20.01±7.21 vs. 26.59±10.66), Fib (g/L, 4.02±1.26 vs. 5.09±1.43), D-dimer (mg/L, 0.24±0.06 vs. 0.31±0.11) were significantly lower in Xuebijing therapy group, and APACHEII score (15.81±3.47 vs. 17.93±3.05) was obviously lowered (all P<0.05). There was statistical difference of the TT (s) between two groups at 3 days (15.68±1.89 vs. 14.65±1.33,P<0.05). There was a significant positive correlation between NF-ΚB DNA binding activity and the levels of TNF-α (r(1)=0.373, r(2)=0.362, r(3)=0.419), PCT (r (1)=0.800, r(2)=0.716, r(3)=0.920) or CRP (r(1)=0.368, r(2)=0.441, r(3)=0.366, all P<0.05) before and 3 days and 7 days after the treatment., Conclusion: NF-ΚB activation and coagulopathy were observed in patients with severe pneumonia, and NF-ΚB was involved in the process of inflammatory response. Inflammatory response was partly alleviated by Xuebijing injection. These effects of Xuebijing injection may be mediated by inhibition of the activation of NF-ΚB and its anticoagulation property.

Objective: To establish a miniSTR multiplex set including three STR loci unlinked from the CODIS loci: D1S1676, D6S1274 and D17S1299, to generate amplified fragment less than 115 bp in size and to study the genotype of degraded DNA samples., Methods: After amplification with different fluorescence labeled primers, the amplified products from 100 unrelated individual and 2 highly degraded specimens were analyzed by 310 Genetic Analyzer., Results: Three miniSTR loci were determined by fluorescence-labeled multiplex-PCR technique. Each locus was successfully genotyped in all 100 samples. In D1S1676, D6S1274 and D17S1299 loci, 9, 9, 7 alleles and 27, 23, 18 genotypes were observed respectively. The distribution of genotype for three miniSTR loci in Chengdu Han population was in accordance with Hardy-Weinberg equilibrium. The combined exclusion probability and the combined discrimination power of the three STR loci in Chengdu Han population were 0.9991 and 0.9160 respectively., Conclusion: This miniSTR multiplex set could be used in individual identification and paternity test. It also provides a new method in the analysis of degraded DNA sample.

Objective: To investigate DNA degradation of porcine retinal cells by single cell gel electrophoresis (SCGE) for estimation of postmortem interval (PMI)., Methods: Degradation of retinal cells was observed by SCGE at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24h after death respectively, under the environmental conditions of being kept in dark place as well as a controlled temperature of (15 +/- 2) degrees C and humidity of (50 +/- 5)%. The comet pictures were captured by fluorescence microscope and analyzed by single cell gel electrophoresis (IMI 1.0)., Results: From 2h to 24h postmortem, the degree of degradation of retinal DNA increased with the prolongation of PMI. The postmortem regression functions of head DNA%, L(T)/L(H), I(T)/I(H) were y = 92.227-5.188 x + 0.019 x2 + 0.001 x3 (R2 = 0.971), y = 0.035e(0.191x) (R2 = 0.947), y = 0.099e(0.264x) (R2 = 0.955), respectively., Conclusion: The examination of retinal cell DNA degradation by SCGE is useful for estimating PMI.

Objective: To study the change of DNA degradation in nucleolus of mice organs and its relationship with the postmortem interval, and to investigate a new accurate method to estimate the postmortem interval., Methods: Eight parameters of cell nuclei were chosen, including the head DNA level, the tail DNA level, the head radius, the tail length, the tail moment, the Olive moment, the head area and the tail area. The changes of DNA degradation were analyzed in skeletal muscle, myocardium, liver, kidney and brain in mice at different intervals (0-72 h postmortem) by using single-cell gel electrophoresis and fluorescent microscope connected with auto-analysis-image system., Results: The tail DNA level, the tail length, the tail moment, the Olive moment and the tail area showed an increasing tendency. The head DNA level, the head radius and the head area showed a decreasing tendency within 72h postmortem in mice. A quadratic regression equation (P < 0.001) and multiple regression equation of DNA degradation tendency were established (P < 0.000 1)., Conclusion: The regression equations established can be used as a new method for estimating postmortem interval in forensic practice.

To avoid sequencing error resulting from use of apyrase in conventional 4- enzyme pyrosequencing system, a non-apyrase 3-enzyme pyrosequencing system with a better performance of quantitative analysis was established. The method is to immobilize biotinylated DNA template, ATP sulfurylase and luciferase on streptavidin-coated magnetic beads for pyrosequencing. After pyrosequencing, ATP produced from the pyrosequencing reaction and excess dNTPs were removed by magnetic separation technique; another dNTP was then dispensed for sequencing reaction, and the components interfering with the next circle of pyrosequencing reaction were removed by the same way, achieving the circular sequencing. This new system can accurately measure base sequences of a target DNA template, and also can quantitatively determine the relative ratio of two alleles. The allele ratios in two SNPs (rs1042917 and rs4818219) having a higher heterozygote rate on chromosome 21 were successfully detected for 16 normal samples and 8 clinical samples from Down's syndrome patients. The results can accurately demonstrate whether or not the target sample has equal copies of chromosome 21 from mother and father. This paper established a non-apyrase 3-enzyme pyrosequencing method, which owns a good perform-ance of quantitative analysis. The method is especially suitable to allelic quantification of an SNP, enabling the rapid diagnosis of Down's syndrome by analyzing allele ratio of SNPs on chromosome 21.

HPV-2 is a very common type of HPV which causes common warts. The E2 protein of virus can repress the activity of the viral early promoter through binding to the specific binding sites in viral LCR. Previously we reported that the repression of a mutated E2 protein of HPV-2 isolated from a patient with huge common wart on the viral early promoter was obviously decreased, and A338V mutation located at the C terminal DNA binding region of E2 protein. In this study, we expressed and purified the recombinant mutated and prototype E2 fusion proteins, both in the contexts of the C terminal and the full length, by prokaryotic expression system. The electrophoretic mobility shift assay showed E2 protein could bind to double-stranded DNA oligos labeled with biotin that covered two E2 binding sites. The DNA binding abilities of both C terminal and full-length mutated E2 proteins were stronger than the prototype analogs. This result indicates that the enhancement of the mutated E2 DNA binding ability may be the molecular mechanism for its impact on the activity of viral promoter, which correlates with the phenotype of extensive common wart.

Aim: To investigate the effect of Nivalenol(NIV) and Selenium(Se) on the levels of IL-1beta and TNF-alpha in the cultured chondrocytes., Methods: Human chondrocytes cultured in vitro were treated with or without NIV and Se. The morphology of chondrocytes was observed by optic microscope. The DNA content was determined by UV Spectrophotometry. The levels of IL-1beta and TNF-alpha in cultured medium were measured by enzyme-linked immunosorbent assay (ELISA)., Results: Hematoxylin & eosin staining indicated there was cell necrosis in the cartilage reconstructed in vitro from both NIV group and NIV+Se group. Compared with the group of NIV toxin, the damage of chondrocytes was less severe when Se was added. NIV could inhibit chondrocyte DNA synthesis. The content of DNA with NIV was lowest than that in other groups. The levels of IL-1beta and TNF-alpha with NIV were significantly higher than control group (P<0.05). After Se was added, the levels did not change significantly compared with the groups without Se., Conclusion: NIV toxin could superinduce IL-1beta and TNF-alpha secretion in chondrocytes, which may be the key mechanism of chondrocyte injury by NIV. Se can partially alleviate the effects of NIV on chondrocytes cultured in vitro.

Chromatin immunoprecipitation (ChIP) is an effective technique to analyze the interactions of DNA binding proteins with the genome in vivo. ChIP coupled with high density microarray (ChIP-chip) or high-throughput sequencing (ChIP-Seq) has generated large amount of data and expected to allow the development of a network describing the cellular transcriptional regulation. Here, we reviewed the ChIP, ChIP-chip, and ChIP-Seq techniques as well as their perspectives. Focus is given to data analysis of ChIP-Seq and the applications of ChIP-chip and ChIP-Seq.

Objective: To investigate the level of hypermethylated ras association domain family 1A (RASSF1A) gene in maternal plasma of pre-eclampsia and its clinical value., Methods: Sixty pre-eclampsia women including 30 mild and 30 severe cases were selected, 60 women with normal pregnancy were studied as control. Free DNA from plasma samples was extracted, fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the concentrations of RASSF1A gene before and after methylation-sensitive restriction digestion. Meanwhile, beta-actin gene was detected as a control to confirm complete enzyme digestion., Results: The median concentration of hypermethylated RASSF1A gene was 3.31-fold higher in samples from pre-eclamptic pregnancies than that in controls. There was significant difference between the mild and severe pre-eclamptic subjects (P<0.05), with the median concentrations of 1659 copies/mL and 2036.50 copies/mL, respectively., Conclusion: Hypermethylated RASSF1A gene in pre-eclampsia plasma was significantly increased and the concentrations were related to the severity of pre-eclampsia.

Protocatechuic acid (P) and veratric acid (V) are phenolic acidic compounds and have a wide biological and pharmaceutical activities, and their interaction with biomacromolecule has been a hot topic. The interaction mechanism of P and V with fsDNA was investigated by fluorescence and UV absorption spectroscopic methods. The UV results showed that P and V have three strong absorption bands at 190-230 nm (K band), 230-270 nm (B band) and 270-310 nm (R band) respectively. When the excitation wavelength was 280 nm, the fluorescence emission bands of P and V were at 338 and 334 nm, respectively, while the fluorescence emission band of DNA was very weak and had little influence on those of the P and V. The fluorescence intensities of the P and V were strongly quenched after interacting with fsDNA, and their Stern-Volmer quenching rate constants were 1.03 x 10(12) and 0.61 x 10(12) L x mol(-1) x s(-1), respectively. It was illustrated that the fluorescence quenching was mainly static and the complex was formed between the drug and fsDNA. When the concentration of DNA was high, their Stern-Volmer curves were not linear, and it was indicated that the quenching mechanism was complex and may contain dynamic quenching process. Their binding constants were calculated based on the static fluorescence quenching, with K(fsDNA/P) = 6.22 x 10(6) L x mol(-1) and K(fsDNA/v) = 1.57 x 10(4) L x mol(-1). The investigation showed that the molecular ratio of V-fsDNA was 1 : 1, while that of P-fsDNA was 1: 2. It was demonstrated that the protocatechuic acid can bind with two bases of fsDNA, which was related to the two hydroxyl groups on the drug molecule. The results showed that the structure of P and V greatly influenced their binding mode with DNA molecules.

Objective: To study the effect of apoptosis induction of pianzihuang in human osteosarcoma U-20S cells., Methods: Sixth SD rats were randomly divided into group A (8 rats) and group B (8 rats) in the study. Among them, 8 rats (group A) were treated with PBS and 8 rats (group B) treated with pianzihuang. (2) Human osteosarcoma U-2OS cells were cultured in nutrient solution (including fetal calf serum of 10%, penicillin of 100 IU/ml, streptomycin of 100 microg/ml), the environmental condition in saturated humidity 5% CO2, 37 degrees C. The osteosarcoma U-2OS cell proliferation effect by different saturation of blood serum was measured by MTT method. And the optimal concentration of the serum (including Pianzihuang) could be obtained. (3) The morphous of osteosarcoma U-2OS cell were observed by inverted phase contrast microscope and electron microscope. (4) Apoptosis cell was detected by TUNEL in situ end labeling. (5) Extracted DNA,agarose gel electrophoresis (AGE) to detect apoptosis cell by transmission electron microscope (TEM)., Results: (1) It was best that osteosarcoma U-2OS cell proliferation effect by 20% of the concentration of the serum (including Pianzihuang). (2) On inverted phase contrast microscope showed that the cells of group A were fusiform adherence growth; the cyton transform shrind and circle in 20% of the concentration of the serum (including Pianzihuang). Follow lasting time,the cell turning circle gradually increased. Necrosis cell and fragment were found by microscope. (3) The detected result of apoptosis cell in 20% of the concentration of the serum(including Pianzihuang) was positive by TUNEL assays. (4) AGE showed "ladder" strand of DNA, a special phenomenon of cellular apoptosis. (5) Chromatin aggregated along corrugativus caryotheca and apoptotic body appeared on cell surface by observation of TEM., Conclusion: Pianzihuang can inhibit proliferation and induce apoptosis for osteosarcoma U-2OS cell.

Objective: To investigate the Change of DNA content (apoptosis rate), mitochondrial transmembrane potential (DeltaPsim) and calcium (Ca(2+)) of rabbit retinal pigment epithelial (RPE) cells cultured with curcumin., Methods: It was an experimental study. The RPE cells were dissociated from rabbit eyes and cultured. The RPE cells in the 4(th) passage were divided into 2 groups: curcumin group and control group (10% FBS-EMDM contains 0.05% dimethyl sulfoxide). The curcumin group contained 3 mass concentration: 10 mg/L, 15 mg/L and 20 mg/L. The MTT assay was used to evaluate the inhibition effect of RPE cells cultured with curcumin after 24 h, 48 h, 72 h and 96 h respectively. The IC(50) value in 24 h, 48 h, 72 h and 96 h were gotten by Linear Regression. Flow cytometry was performed to detect the change of DNA content (apoptosis rate), DeltaPsim and Ca(2+) of RPE cells cultured with curcumin (15 mg/L) after 8 h, 24 h, 48 h and 72 h respectively., Results: RPE cells were significantly inhibited by curcumin in a dose dependent and time dependent manner. The IC(50) value of curcumin at 24 h, 48 h, 72 h and 96 h was 29.31 mg/L, 17.50 mg/L, 13.24 mg/L and 10.99 mg/L respectively. Ca(2+) was significantly increased at 8 h, 24 h, 48 h and 72 h after cultured with curcumin (15 mg/L) than that of the control group respectively (t = 7.50, 10.61, 20.74, 21.14, P < 0.01), and DeltaPsim was significantly decreased at 8 h, 24 h, 48 h and 72 h after cultured with curcumin (15 mg/L) than that of the control group respectively (t = 7.50, 11.74, 14.91, 15.29, P < 0.01). There was no change of DNA content in RPE cells at 8h after cultured with curcumin (15 mg/L), but significantly lower than that of the control group at 24 h, 48 h and 72 h respectively (t = 10.00, 14.68, 13.68, P < 0.01)., Conclusion: The apoptosis of RPE cells induced by curcumin is caused by increase of Ca(2+) and decrease of DeltaPsim that causes decrease of DNA content. The RPE cells are significantly inhibited by curcumin, which may become a potential drug to prevent and treat proliferative vitreoretinopathy.

Investigation of DNA-protein interactions is fundamental to understand the mechanism underlying a variety of life processes. In this article, various types of biochemical methods in DNA-protein interaction study in vivo and in vitro at the level of DNA, protein, and the complex, respectively were briefly reviewed. Traditional assays including Nitrocellulose filter-binding assay, Footprinting, EMSA, and Southwestern blotting were summarized. In addition, chromatin immunoprecipitation techniques including nChIP, xChIP, and ChIP-on-chip, which were widely used in epigenetics, were particularly introduced.

Cobalt (II) polyamidomine dendrimer was prepared by the reaction of cobalt chloride, glyoxal and polyamidomine dendrimer of 5.0 generation. The interaction of cobalt (II) polyamidomine dendrimer complex with herring sperm (hsDNA) was carried out using methylene blue (MB) as the probe molecule by absorption and fluorescence spectroscopy and synchronous fluorescence spectroscopy. The results showed that the intensity of absorption peaks and fluorescence peaks increased when the complex interacted with hsDNA. The effect of sodium chloride showed that sodium ion can significantly constrain the interaction of cobalt(II) polyamidomine dendrimer with hsDNA. The curves indicated the competitive inhibition of MB binding to hsDNA in the presence of cobalt (II) polyamidomine dendrimer complexes, also MB could insert into interior of cobalt (II) polyamidomine dendrimer complexes. The results suggested that the complex mainly interacted with negatively charged phosphate moieties on hsDNA through electrostatic attraction and stacked on the surface of double stranded hsDNA, which may reduce the binding affinity of MB to hsDNA in the surrounding site. It was indicated that sodium ion might neutralize the negatively charged phosphate backbone of hsDNA, and then weaken the electrostatic attraction between complexes and hsDNA.

Interest in the interaction between cationic porphyrins, particularly derivatives of meso-tetra(N-methylpyridinium-4-yl) porphyrin(TMPyP), and DNA abounds because they are versatile DNA-binding agents that could find application in photodynamic therapy, cancer detection, artificial nucleases, virus inhibition and so on. The interaction of two water-soluble cationic porphyrins, meso-tetrakis(4-N, N, N-trimethylanilinium) porphyrin (TMAP) and 5-phenyl-10,15,20-tris[4-(N-methyl) pyridinium]porphyrin (TriMPyP), with calf thymus DNA (ctDNA) was studied by UV-Vis absorption spectroscopy, fluorescence spectroscopy and resonance light scattering technique. TriMPyP forms aggregate in water due to the molecular asymmetry while TMAP exists as monomers. At lower concentrations of ctDNA (R > 1, R = c(TMAP)/c(DNA) base pair), the interaction of TMAP with DNA leads to significant hypochromicity and bathochromic shift of absorption spectra. And the fluorescence of TMAP was quenched while it showed enhanced resonance light scattering signals. But the extent of enhancement of resonance light scattering signals is very small, so the aggregate of TMAP is not very high. These observations indicate the self-stacking of TMAP along the DNA surface. At higher concentrations of ctDNA (R < 1), TMAP association with DNA is via outside binding which is accompanied with hyperchromic effect and fluorescence enhancement while the resonance light scattering signals is reduced. DNA addition decreases the fluorescence intensity of TriMPyP and it shifts the peak to the higher wavelengths (red shift). The interaction with DNA promotes the aggregation of TriMPyP and no simple outside binding is observed even at higher concentrations of ctDNA. The steric effect of molecular distortion constrains the intercalation or further binding to DNA. The effect of ionic strength on the interaction was investigated at two DNA concentrations, 1.2 and 24.0 micromol x L(-1), for TMAP. The Interactions of both porphyrins with DNA show high sensitivity to ionic strength. By addition of NaCl, electrostatic attraction is decreased, resulting in the change of binding mode.

A new nonviral gene vector--urocanic acid-coupled chitosan (UAC) was prepared by the reaction of the activated urocanic acid (UA) with the amine group on the chitosan (CTS). The structure of UAC was confirmed with FT-IR, 1H NMR and element analysis. The influencing factors of substitution values were studied by orthogonal test, and the substitution values of UAC increased with the prolongation of activating time of UA and the increasing ratio of UA to CTS. The condensation ability and the resistance to DNase I of UAC/pDNA were evaluated by agarose gel electrophoresis, and UAC showed good condensation ability with pDNA, well protecting pDNA from the degradation by DNase I. The particle size and zeta potential were evaluated by zetasizer, and the results showed that the UAC/pDNA complex was well stable and could easily enter into cells. The transfection studies were performed with HepG2 cells in vitro. It showed that the in vitro transfection of UAC/pDNA was efficient in HepG2 cells and could express more green fluorescent proteins than that of CTS. So the UAC is easy to prepare and a promising non-viral gene vector.

Objective: To detect the most prevalent mutations, R778L and P992L of ATP8B gene, in Chinese Wilson disease(WD) patients by high resolution melting (HRM) analysis after polymerase chain reaction (PCR)., Methods: Genomic DNA was extracted from peripheral blood samples obtained from 30 cases of WD by the standard phenol/chloroform method. DNA fragments encompassing ATP7B exons 8 and 13 were produced by PCR amplification. The amplicons containing the R778L or P992L mutations were then generated by nested PCR. The nested PCR products were subjected to HRM analysis using the HR-1 instrument. Mutations detected in HRM analysis were verified by restriction analysis using restriction enzyme (MspI or AluI or AfaI) or DNA sequencing., Results: HRM analysis of the fragments encompassing ATP7B exon 8 showed four curve patterns. Subsequent restriction analysis and DNA sequencing proved that the four different curves represent four different genotypes: the wild type, the R778L/R778L homozygote, the R778L heterozygote, and the R778L/752.33delG compound heterozygote. Three HRM curve patterns were observed for the fragments encompassing ATP7B exon 13, representing the wild type, the P992L heterozygote, and the P992L/S975Y compound heterozygote. In our studied samples, allele frequencies of the R778L, P992L and S975Y mutations were 25%, 15% and 1.67%, respectively., Conclusion: HRM analysis is a simple, accurate and sensitive approach for rapid detection of the ATP7B mutations and could be used as an optimized method for genetic testing in WD.

Background & Objective: Radiation pulmonary fibrosis (RPF) is characterized by fibroblast proliferation and excessive accumulation of extracellular matrix (ECM). Transforming growth factor beta (TGFbeta) is a switch factor in the initiation and development of RPF and serves as a therapeutic target. Blocking TGFbeta1 signal transduction pathway might alleviate RPF. This study was to investigate the effects of two Smad pathway inhibitors, SB203580 and WP631, on Smad signal transduction pathway in human lung fibroblasts (HLFs) after irradiation., Methods: HLFs were pretreated with 25 micromol/L SB203580 or 5 nmol/L WP631, then irradiated with 3 Gy 60Co gamma rays and stimulated by 10 microg/L TGFbeta1. The transcriptional activity of SP1 and AP1 were measured using electrophoretic mobility shift assay (EMSA). Expressions of Smad3, Smad4, Smad7, p-Smad3 and P21(WAF1/CIP1) were detected by Western blot. The expression of type Iplasminogen activator inhibitor (PAI-I) was detected by immunohistochemical staining The cell cycle was measured by FACS., Results: After irradiation. with 3 Gy gamma rays and stimulation by TGFbeta1, HLFs pre-incubated with SB203580 or WP631 were increased in G2-M phase and decreased in S phase as compared with cells without pretreatment. p21(WAF1/CIP1) and p-Smad3 were decreased in HLFs pretreated with SB203580, while PAI-1 was decreased in HLFs pretreated with WP631. Furthermore, the transcriptional activity of SP1 and AP1 was inhibited by WP631., Conclusions: SB203580 and WP631 can abrogate excessive proliferation, expressions of p21(WAF1/CIP1) and PAI-1 induced by gamma rays and TGFbeta1 in HLFs through blocking Smad signal transduction pathway.

Free circulating DNA is one kind of extracellular free DNA, which was examined in the zoology, botany and the human's body fluid. The detection of free circulating DNA had already been applied in monitoring the disease, embryo's pre-natal diagnosis, tumor research. This article reviewed some biology characteristics of free circulating DNA, the recent researches of free circulating DNA and its application.

Cell/gene-activated biomaterials are the main tendency and characteristics of the biomaterial researches for the future. The gene delivery based on the polymeric materials as vectors has potential applications in gene therapy, regenerative medicine and tissue engineering. In this paper, the application of layer-by-layer (LBL) technique in advanced biomaterial surface engineering is reviewed and its potential application in the construction of in situ gene delivery system onto biomaterial surface is in turn addressed. These are of great significance to biomaterial researches.

Objective: To investigate the changes in the activity of nuclear factor-kappaB (NF-kappa B) in mice with dextran sulphate sodium (DSS)-induced rat colitis and its modulalorg effect on intercellular adhesion molecule-1 (ICAM-1) expression., Methods: Twenty normal male mice were randomized into DSS group and normal saline (NS) control group according to a matched-pair design. From days 1 to 7, the mice in DSS group were subjected to oral administration of 5%DSS solution, and from days 8 to 20, NS was given instead, for a total of 3 cycles. In the control group, only NS was administered. The colonic pathology was observed using HE staining and the mucosa 1 damage was scored for each mouse. The DNA-binding activity of NF-kappa B was tested by electrophoretic mobility shift assay, and the expressions of ICAM-1 and NF-kappa B p65 were detected using immunohistochemistry., Results: The DNA-binding activity of NF-kappa B was significantly increased in DSS group as compared with NS group. ICAM-1 and p65 expressions were detected in the nuclei of the vascular endothelial and inflammatory cells, especially in the mucosa and submucosa, but such positive cells were seldom observed in NS group. A positive correlation was found between the DNA-binding activity of NF-kappa B and ICAM-1 expression., Conclusion: NF-kappa B activation is an important event in the development of DSS-induced colitis in that activated NF-kappa B upregulates ICAM-1 expression during colonic inflammation.

Objective: To study the effect of total glucosides of paeony (TGP) on lipopolysaccharides (LPS)-induced nuclear factor-kappaB (NF-kappaB) activation in macrophages., Method: Rat peritoneal macrophages were pre-treated with TGP for 2 h and stimulated with LPS for 20 min or 0.5 h. Inhibitory kappaBalpha (IkappaBalpha) protein in the cytoplasm and NF-kappaB p65 protein in the nuclear were analyzed by western blot. Further, DNA binding activity of NF-kappaB complex was detected., Result: TGP enhanced the amounts of IkappaBalpha protein in the cytoplasm and decreased the amounts of NF-kappaB p65 protein in the nuclear of LPS-induced macrophages. TGP also inhibited the LPS-mediated DNA binding activity of NF-kappaB complex in macrophages., Conclusion: TGP can inhibit LPS-induced NF-kappaB activation in macrophages through arresting IKBalpha protein degradation, NF-kappaB p65 protein nuclear translocation and DNA binding activity of NF-kappaB complex.

Objective: To explore the effects of DNA-protein crosslinks (DPC) of liver, kidney and spermery cell induced by microcystin-LR (MR-LR)., Methods: Kunming male mice were treated by eritoneal injection with different doses of MC-LR. The quantities of junction DNA and free DNA of liver, kidney, testicle cell were detected, and the DPC coefficient were calculated, then we can judge the degree of DNA and protein crosslinks. The DPC coefficient equal junction DNA/(junction DNA + free DNA)., Results: Of the groups treated with MC-LR, DPC formations in liver cell were observed significantly increased, in comparison it on with the control groups (P < 0.05). DPC formations in kidney cell were observed significantly increased at the doses of 3 microg/kg bw and 6 microg/kg bw MC-LR, in comparison it on with the control groups (P < 0.05), but there was no effect at the dose of 12 microg/kg bw MC-LR, in comparison it on with the control groups (P > 0.05). At the dose of 3 microg/kg bw MC-LR, DPC formation in testicle cell were not observed significantly increased, in compare it on with the control groups (P > 0.05). DPC formation in testicle cell were observed significantly increased at the dose of 6 microg/kg bw and 12 microg/kg bw MC-LR, in compare it on with the control groups (P < 0.05)., Conclusion: Microcystin-LR could induce DPC formation in liver, kidney and testicle cells of male mice.

To synthesize aristolochic acid (AA)-2'-deoxyguanosine 5'-monophosphate (dGp) adducts in vitro and develop a novel method for the characterization of the adducts using multiple mass spectrometric techniques. AA was incubated with dGp in vitro using either enzymatic activation (by xanthine oxidase) or chemical activation (by zinc) to synthesize AA-dGp adducts, and the reaction conditions were optimized. Crude extracts were analyzed by techniques of liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-MS/MS) and high accuracy mass data and isotope pattern of super high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICRMS). The quasi-molecular ion peaks of the AA-dGp adducts were obtained in the negative ion mode. Analysis by electrospray ionization/tandem mass spectrometry (ESI-MS/MS) provided useful structural information about AA-dGp adducts. AA can bind covalently to the exocyclic amino group of deoxyguanosine to form AA-dGp adducts. MS analysis is a powerful tool to detect and identify AA-dGp adducts simply, rapidly and accurately.