Preparation and Characterization of Aptamers Against O,p'-DDT.

The compound 1,1,1-trichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl) ethane (o,p'-DDT) has been identified as one of the endocrine-disrupting chemicals causing adverse effects on wildlife and even humans through bioaccumulation. Its detection has become increasingly important. We have obtained candidate aptamers binding to o,p'-DDT by a systematic evolution of ligands by exponential enrichment (SELEX) protocol. Five out of seventeen candidate sequences were selected for preliminary characterization by SYBR Green I assay. One sequence with highest fluorescence response with o,p'-DDT, designated DDT_13, was chosen for further characterization. Its dissociation constant (Kd) was determined to be 412.3 ± 124.6 nM. DDT_13 exhibited low cross-binding activities on other tested small molecules. The good bioactivities of DDT_13 were demonstrated for the analysis of spiked lake water and tap water samples. This study provides a novel o,p'-DDT-specific probe for its future applications.

[Advances in the application of gene therapy for Parkinson's disease with adeno-associated virus].

Vectors used to carry foreign genes play an important role in gene therapy, among which, the adeno-associated virus (AAV) has many advantages, such as nonpathogenicity, low immunogenicity, stable and long-term expression and multiple-tissue-type infection, etc. These advantages have made AAV one of the most potential vectors in gene therapy, and widely used in many clinical researches, for example, Parkinson's disease. This paper introduces the biological characteristics of AAV and the latest research progress of AAV carrying neurotrophic factor, dopamine synthesis related enzymes and glutamic acid decarboxylase gene in the gene therapy of Parkinson's disease.

[A preliminary study of anti-aging and wound healing of recombination cytoglobin].

In this paper, the preliminary study on antioxidant, enhancement of antioxidant enzymes activity, reducing the content of oxygen free radicals, delaying skin aging of the recombination cytoglobin (rCygb) purified by our lab were investigated through human keratinocyte cell line (HaCAT) H2O2 oxidative stress model, mouse skin aging model caused by continuous subcutaneous injection D-gal, rat acute liver injury model induced by CCl4 and rat skin wound healing model. The results showed that rCygb improved the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), reduced the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) as well as decreased the content of malondialdehyde (MDA). Skin biopsy showed that rCygb promoted angiogenesis, increased expression of collagen and improved the anti-inflammatory ability. All results displayed that rCygb improved the oxygen free radical scavenging ability, delayed skin aging and promoted wound healing.

Polymer-capped CdSe/ZnS quantum dots for the sensitive detection of Cu2+ and Hg2+ and the quenching mechanism.

In this work, poly(styrene-co-maleic anhydride)-capped CdSe/ZnS quantum dots (QDs) aminolyzed with ethanolamine are proposed as fluorescent probes for the detection of Cu2+ and Hg2+, and two different quenching mechanisms are discussed in detail. The coordination abilities of the surface polymer of CdSe/ZnS QDs and two metal ions are calculated by density functional theory (DFT). The photoinduced electron transfer from excited QDs to Cu2+ unoccupied orbitals is enhanced due to the coordination between Cu2+ and the surface polymer of QDs. The electron transfer consumes non-radiative energy and performs fluorescence quenching. For Hg2+, the formation of HgS and the slight aggregation of polymer-coated CdSe/ZnS QDs lead to fluorescence quenching. The probe is sensitive to both Cu2+ and Hg2+, and the response can be detected within 1 min without adjusting the pH. With the addition of a masking agent, Cu2+ and Hg2+ can be exclusively detected in coexistence with another ion. For Cu2+, a linear relation in the concentration ranging from 0.02 to 0.7 µM was found between the relative fluorescence intensity (F0/F) and the concentration of Cu2+; the limit of detection (S/N = 3) is 6.94 nM. For Hg2+, a linear relation ranging from 0.1 to 1.4 µM was found between ln(F0/F) and the concentration of Hg2+; the limit of detection is 20.58 nM.

Cell surface expression of nucleolin mediates the antiangiogenic and antitumor activities of kallistatin.

Kallistatin is a unique serine proteinase inhibitor and heparin-binding protein. A previous study conducted by our group indicated that kallistatin has antiangiogenic and antitumoral activities. In the present study, we report that kallistatin specifically binds to membrane surface-expressed nucleolin with high affinity. Antibody-mediated neutralization or siRNA-induced nucleolin knockdown results in loss of kallistatin suppression of endothelial cell proliferation and migration in vitro and tumor angiogenesis and growth in vivo. In addition, we show that kallistatin is internalized and transported into cell nuclei of endothelial cells via nucleolin. Within the nucleus, kallistatin inhibits the phosphorylation of nucleolin, which is a critical step required for cell proliferation. Thus, we demonstrate that nucleolin is a novel functional receptor of kallistatin that mediates its antiangiogenic and antitumor activities. These findings provide mechanistic insights into the inhibitory effects of kallistatin on endothelial cell growth, tumor cell proliferation, and tumor-related angiogenesis.

Kallistatin protects against bleomycin-induced idiopathic pulmonary fibrosis by inhibiting angiogenesis and inflammation.

Aberrant angiogenesis and vascular remodeling are the main features of idiopathic pulmonary fibrosis. Kallistatin is an anti-angiogenic peptide with known effects on endothelial cells. This study aimed to demonstrate that kallistatin has beneficial effects on bleomycin (BLM)-induced pulmonary fibrosis in a rat model by inhibiting angiogenesis. Twenty-five rats were randomly divided into five experimental groups: (A) Saline only (SA)-as the negative control, (B) BLM only (BLM)-as the model group, (C) BLM and 0.1 mg/kg kallistatin (L-Kal), (D) BLM and 0.5 mg/kg kallistatin (M-Kal), and (E) BLM and 2.5 mg/kg kallistatin (H-Kal). Fibrillar collagen was quantified by Masson's trichrome and hematoxylin-eosin staining. Transforming growth factor-ß1 (TGF-ß1), α-smooth-muscle-actin (α-SMA) and microvascular density (MVD) were measured by immunohistochemistry. Vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), and tumor necrosis factor-α (TNF-α) were assayed by Western immunoblotting or ELISA. Daily administration of kallistatin attenuated fibrosis in BLM-induced pulmonary fibrosis, as shown by histology. During inflammation from BLM-induced pulmonary fibrosis, kallistatin reduced the number of inflammatory cells infiltrating the bronchoalveolar lavage fluid. Kallistatin also inhibited VEGF expression and phosphorylation of VEGFR2 (Flk-1). In vitro, kallistatin blocked tube formation by inhibiting Flk-1 and GSK-3ß phosphorylation. The results demonstrated that continuous administration of kallistatin attenuated BLM-induced pulmonary fibrosis and improved survival of BLM rats. Reducing pulmonary fibrosis was achieved by partial inhibition of pulmonary inflammation and angiogenesis.

[Relationship between hepatitis B virus genotype, BCP/Pre-C region mutations and risk of hepatocellular carcinoma in Guangxi Zhuang Autonomous Region].

OBJECTIVE: To investigate the relationship between hepatitis B virus (HBV) genotype, the mutation in basic core promoter (BCP) region/pre-core (Pre-C) region and the incidence of hepatocellular carcinoma (HCC) in Fusui county of Guangxi Zhuang Autonomous Region (Guangxi), a area with high incidence of HCC. METHODS: In this case-control study, 53 HCC patients and 70 asymptomatic HBV carriers were enrolled. Blood samples were collected from them for serum separation and HBV DNA extraction. The DNA sequences of the S region and BCP/Pre-C region of HBV was determined by direct sequencing following nested-PCR amplification. The relationship between the genotype, gene mutation of HBV and the incidence of HCC was analyzed. RESULTS: The mutation rates of the A1762T/G1764A in the BCP region and the T1858C in the Pre-C region of HBV were significantly higher in HCC group than in control group (94.3% vs. 75.7%, P = 0.006; 50.9% vs. 31.4%, P = 0.029). The mutation rate of A1775G was significantly higher in control group (28.6%) than in HCC group (13.2%) (P = 0.041). Multiple logistic regression analysis indicated that A1762T/G1764A and T1858C mutations are the risk factors for the development of HCC (OR = 5.459, 95% CI: 1.397-21.332, P = 0.015; OR = 3.881, 95% CI: 1.462-10.305, P = 0.006). A1775G is the protective factor in the development of HCC (OR = 0.192, 95% CI: 0.059-0.622, P = 0.006). CONCLUSION: The present investigation showed that BCP A1762T/G1764A, A1775G and Pre-C T1858C mutations are correlated with the incidence of HCC in Fusui county of Guangxi.

microRNA-133: expression, function and therapeutic potential in muscle diseases and cancer.

microRNAs (miRNAs) are a class of small non-coding RNAs that are 18-25 nucleotides (nt) in length and negatively regulate gene expression post-transcriptionally. miRNAs are known to mediate myriad processes and pathways. While many miRNAs are expressed ubiquitously, some are expressed in a tissue specific manner. miR-133 is one of the most studied and best characterized miRNAs to date. Specifically expressed in muscles, it has been classified as myomiRNAs and is necessary for proper skeletal and cardiac muscle development and function. Genes encoding miR-133 (miR-133a-1, miR-133a-2 and miR-133b) are transcribed as bicistronic transcripts together with miR-1-2, miR-1-1, and miR-206, respectively. However, they exhibit opposing impacts on muscle development. miR-133 gets involved in muscle development by targeting a lot of genes, including SFR, HDAC4, cyclin D2 and so on. Its aberrant expression has been linked to many diseases in skeletal muscle and cardiac muscle such as cardiac hypertrophy, muscular dystrophy, heart failure, cardiac arrhythmia. Beyond the study in muscle, miR-133 has been implicated in cancer and identified as a key factor in cancer development, including bladder cancer, prostate cancer and so on. Much more attention has been drawn to the versatile molecular functions of miR-133, making it a truly valuable therapeutic gene in miRNA-based gene therapy. In this review, we identified and summarized the results of studies of miR-133 with emphasis on its function in human diseases in muscle and cancer, and highlighted its therapeutic value. It might provide researchers a new insight into the biological significance of miR-133.

[Expression, purification, and characterization of fusion protein TAT-cytoglobin].

he aim of this study was to obtain a cell-penetrating cytoglobin (Cygb), which combines the transmembrane function of cell-penetrating peptides TAT with the anti-aging and anti-fibrotic role of cytoglobin. The Cygb gene was complexed with TAT gene by overlapping PCR, inserted into the vector pET22b to construct the recombinant expression plasmid (pET22b-TAT-Cygb) and then transformed into Escherichia coli BL21 (DE3). The fusion protein TAT-Cygb, whose expression was induced by lactose, was purified by CM Sepharose Fast Flow Protocol and verified by Western blotting. The final TAT-Cygb had a molecular weight of 23 kDa with 95% purity, as shown by SDS-PAGE. As demonstrated by bioactivity experiments, TAT-Cygb exhibited a high specific peroxidase activity up to (422.30 ± 0.36) U/mg. Both TAT-Cygb and Cygb pretreatment group could protect Hacat cells against oxidation of H2O2, but only TAT-Cygb treatment group could remedy cells injuried by H2O2 (RGR = 98%), which was significantly different from Cygb treatment group (RGR = 79%). We successfully obtained the bioactive and cell-penetrating fusion protein TAT-Cygb that has the potential application in anti-aging, anti-fibrotic and anti-cancer.

[Protective effects of PEG modified recombinant cytoglobin on acute liver injury in mice].

To investigate the protective effect of polyethylene glycol (PEG) modified recombinant cytoglobin (PEG-rCygb) on acute liver damage in mice. The acute liver injury model of KM mice was induced by CCl4 and then treated with PEG-rCygb, The liver and blood samples were collected for biochemical and histopathological analysis. The results showed that PEG-rCygb reduced the liver mass index and decreased significantly the levels of alanine amiotransferase (AST) and aspartate transaminase (ALT) in mouse serum. In liver tissues, the content of malondialdehyde (MDA) was decreased, whereas the content of glutathione (GSH) was increased in PEG-rCygb treated group. PEG-rCygb also elevated the activities of total super oxidedismutase (T-SOD) and catalase (CAT) in liver tissues. HE staining of liver tissue slices revealed that PEG-rCygb relieved fatty degeneration of liver, decreased inflammatory factors and reduced liver cell injury. Further in vitro experiments indicated that the protective effects of PEG-rCygb on hepatic stellate cell (HSC) against H2O2 were enhanced compared with that of rCygb. All results indicated that the PEG-rCygb promoted oxygen free radical scavenging ability and prevented acute liver injury in KM mice induced by CCl4.

Double-stranded Let-7 mimics, potential candidates for cancer gene therapy.

MicroRNAs (miRNAs), a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The ability of one single miRNA regulating multiple functionally related mRNAs makes it a new potential candidate for cancer gene therapy. Let-7s miRNAs have been demonstrated as tumor-suppressor genes in various types of cancers, providing one choice of gene therapy by replenishing this miRNA. In the present studies, we demonstrate that the chemically synthesized, double-stranded Let-7 mimics can inhibit the growth and migration and induce the cell cycle arrest of lung cancer cell lines in vitro. Let-7 mimics silence gene expression by binding to the 3' UTR of targeting mRNAs. Mutation of seed sequence significantly depresses the gene silencing activity of Let-7 mimics. Our results also demonstrate that it is possible to increase the activity of Let-7s through mutating the sequence within the 3'end of the antisense strand. Directly, co-transfection Let-7 mimics with active siRNAs impairs the anti-cancer activities of Let-7 mimics. However, a 3-h interval between the introduction of Let-7 mimics and a kind of siRNA avoids the competition and enhances the anti-cancer activities of Let-7 mimics. Taken together, these results have revealed that Let-7s mimics are potential candidates for cancer gene therapy.

[Role of cytoglobin in protecting hepatic stellate cells against oxidation induced damage].

The aim of this study was to reveal the protection role and the related mechanism of cytoglobin on the oxidation induced hepatic stellate cell damage. We applied siRNA to interfere the endogenous cytoglobin gene, used recombinant cytoglobin protein to treat the completely activated human hepatic stellate cell line LX-2 and the incompletely activated primary rat hepatic stellate cells, or over-expressed cytoglobin protein in LX-2 cells. We used two different oxidative-stress related models, the hydrogen peroxide model and the iron-overload model in our experiments and investigated the proliferation status and the intracellular superoxide level of the cells. The results showed that endogenous cytoglobin exerted significant protective effects on hydrogen peroxide or iron-overload induced LX-2 cell damage, confirming that upregulation of cytoglobin was the protective response of activated hepatic stellate cells to oxidative stress. Recombinant cytoglobin protein could protect LX-2 cells from oxidation induced damage, and prevent primary rat hepatic stellate cells from excessive proliferation and injury. The cytoplasmic reactive oxygen species (ROS) scavenging capacity of the recombinant cytoglobin protein was not as good as its capacity in scavenging ROS outside the cells, likely owing to the lack of active transporting mechanisms. Intracellular over-expression of cytoglobin protein could exert significant protective effect on LX-2 cells treated with hydrogen peroxide or iron-overload. Our results would accelerate the exploitation of new anti-fibrotic targets.

[Capsid assembly and DNA encapsidation of adeno-associated virus].

Recombinant adeno-associated viral vectors (rAAV) have been widely used as gene therapy vectors in clinical trials. Here, we reviewed the genomic structures and replication mechanisms of wt-AAV. Then, the assembly of capsid and the encapsidation of genomic DNA, two major events during AAV pakaging, was discussed in detail. Although the overall pattern of virus assembly and encapsidation is known, the molecular mechanisms and the structure-function relationship involved in these processes are not well understood. Further elucidatation of these processes may improve the production technology of rAAV and develop gene drug based on rAAV.

[High level expression of recombinant human kallistatin in Pichia pastoris and its bioactivity].

In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.

[Recombinant batroxobin expressed highly in Pichia pastoris].

Methylotrophic yeast, Pichia pastoris was used to express recombinant batroxobin, and a technology route of producing recombinant protein was finally established. We synthesized batroxobin gene artificially by means of recursive PCR. pPIC9-batroxobin was constructed and transformed into Pichia pastoris GS115 (his4). Recombinant batroxobin was expressed in yeast engineering strain and it was purified from the culture supernatant. 10 mg of recombinant batroxobin was purified from 1 liter fermentation media, it exhibited specific activity of 238 NIH units/mg and had molecular weight of 30.55 kD. The purified recombinant protein converted fibrinogen into fibrin clot in vitro, and shortened bleeding time in vivo. This study laid a foundation of development of hemostatic of recombinant snake venom thrombin-like enzyme.

Expression and purification of ancrod, an anticoagulant drug, in Pichia pastoris.

Ancrod is known as a thrombin-like enzyme from the venom of Calloselasma rhodostoma. The cDNA encoding ancrod was synthesized with a yeast bias codon and inserted into the eukaryotic expression vector pPIC9 and was subsequently expressed in the yeast Pichia pastoris. Recombinant ancrod was produced in 5-L bioreactor using a sorbitol-methanol feeding strategy and recovered from the fermentation broth by hydrophobic, affinity, and ion exchange chromatography. SDS-PAGE analysis revealed that ancrod was heterogeneously glycosylated and running at the expected molecular weight of 43-48 kDa which decreased to about 29 kDa after deglycosylation with N-glycosidase F. The fibrinogenolytic and zymographic activity of the recombinant ancrod were determined and were found to be similar to that of the native protein.

Expression of the human phosphatases of regenerating liver (PRLs) in colonic adenocarcinoma and its correlation with lymph node metastasis.

BACKGROUND: Human phosphatases of regenerating liver (PRLs) can induce cell growth, differentiation, and malignant transformation. In this study, we used specific polyclonal antibodies against PRLs to investigate their expression in colonic adenocarcinomas and its correlation with patient gender, age, tumor differentiation, localization, invasion, and metastasis. MATERIALS AND METHODS: The polyclonal antibodies against PRL-1, PRL-2, and PRL-3 were produced and purified. The expression of PRLs in human colorectal carcinoma cell lines (SW480 and SW620) was examined by Western blotting. We also examined their expression in normal and pathologic tissues from the human colon. The tissues included 49 primary colonic adenocarcinomas, 14 cases with lymph node metastases, 15 colonic adenomas, and 12 normal colon samples. Hematoxylin and eosin staining, immunohistochemistry, and semiquantitative morphological analysis were used to evaluate the sections. RESULTS: PRLs were widely expressed in SW480 and SW620. PRL-1, PRL-2, and PRL-3 were expressed, respectively, in 16, 10, and 16% of primary colonic adenocarcinomas. In contrast, PRLs were strongly expressed in all lymph node metastases. There were no significant correlations between the expression of PRLs and patient gender, age, tumor differentiation, depth of invasion, or localization of tumor within the different sections of the colon. PRLs were not expressed in normal colon tissues or in colonic adenomas. PRLs were mainly expressed in the cytoplasm and at the cytoplasmic membranes of the colonic adenocarcinoma cells as well as in the endothelial cells and the surrounding smooth muscle cells of larger vessels in the lymph node metastases. CONCLUSION: Colonic adenocarcinoma cells have the ability to produce PRLs, which may relate to the lymph node metastasis of colonic adenocarcinoma.