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ABSTRACT: To study Hepatitis B virus (HBV) infection and its association with hepatocellular carcinoma (HCC) at the miRNA level.
Three cellular models were used to investigate miRNA expression changes during HBV infection: human HepG2 hepatoblastoma cell line as a model without HBV infection; HepG2 cell line transfected with a 1.3-fold full-length HBV genome as an acute infection model; and HepG2.2.15 cell line, which is derived from HepG2 and stably transfected with a complete HBV genome, as a chronic infection model. The miRNA levels were examined using microarray technology. To explore the relationship between HBV infection and HCC genesis at the miRNA level, we downloaded from national center for biotechnology information Gene Expression Omnibus an miRNA expression dataset derived from HCC patients, most of whom are HBV carriers. We compared the miRNA expression alterations during HBV infection with those in HCC patients, by analyzing miRNA expression change profiles statistically.
Seventy-seven and 48 miRNAs were differentially expressed during acute and chronic HBV infection, respectively. Among these miRNAs, 25 were in common, the intersection of which was significant under the hypergeometric test (P = 1.3 × 10⁻¹¹). Fourteen miRNAs were observed to change coherently in the acute and chronic infections, with one upregulated and 13 downregulated. Eleven showed inverse changes during the two phases of infection; downregulated in the acute infection and upregulated in the chronic infection. The results imply that common and specific mechanisms exist at the miRNA level during acute and chronic HBV infection. Besides, comparative analysis of the miRNA expression changes during HBV infection with those in HCC indicates that, although miRNA expression changes during HBV infection are distinct from those in HCC patients (P < 2.2 × 10⁻¹⁶), they exhibited significant correlations (P = 0.0229 for acute infection; P = 0.0084 for chronic infection). Perturbation of miRNA expression during chronic HBV infection was closer to that in HCC patients than that during acute HBV infection. This observation implies the contribution of miRNAs to HCC genesis from HBV infection. According to their patterns of differential expression in acute and chronic HBV infection, as well as in HCC, miRNAs of potential research interest could be identified, such as miR-18a/miR-18b, miR-106a, miR-221 and miR-101. For instance, the gradient expression alteration of miR-221 in the above three phases, which is downregulated in acute HBV infection, normally expressed in chronic HBV infection, and upregulated in HCC, indicates that it may be a key effector for progression of the disease.
Our analysis provides insights into HBV infection and related HCC in relation to miRNAs, and reveals some candidate miRNAs for future studies.
World Journal of Gastroenterology 07/2011; 17(28):3353-8. · 2.47 Impact Factor
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Pu Hu,
Lei Sun,
Zhi-Qiang Zhu,
Xiao-Wei Hou,
Shu Wang,
Shan-Shan Yu,
Hui-Li Wang,
Ping Zhang,
Ming Wang,
Li-Wen Niu, Mai-Kun Teng,
Di-Yun Ruan
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ABSTRACT: Snake secreted phospholipasesA2 (sPLA2s) are widely used as pharmacological tools to investigate their role in diverse pathophysiological processes. Some members of snake venom sPLA2s have been found to block voltage-activated K(+) channels (K(v) channels). However, most studies involved in their effects on ion channels were indirectly performed on motor nerve terminals while few studies were directly done on native neurons. Here, a novel snake sPLA2 peptide neurotoxin, Natratoxin, composed of 119 amino acid residues and purified from Naja atra venom was reported. It was characterized using whole-cell patch-clamp in acutely dissociated rat dorsal root ganglion (DRG) neurons. It was found to effectively inhibit A-type K(+) currents and cause alterations of channel gating characters, such as the shifts of steady-state activation and inactivation curves to hyperpolarization direction and changes of V(1/2) and slope factor. Therefore, Natratoxin was suggested to be a gating modifier of K(v) channel. In addition, this inhibitory effect was found to be independent of its enzymatic activity. These results suggested that the toxin enacted its inhibitory effect by binding to K(v) channel. To further elucidate the structural basis for this electrophysiological phenomenon, we determined the crystal structure of Natratoxin at 2.2 A resolution by molecular replacement method and refined to an R-factor of 0.190. The observed overall fold has a different structural organization from other K(+) channel inhibitors in animal toxins. Compared with other K(v) channel inhibitors, a similar putative functional surface in its C-terminal was revealed to contribute to protein-protein interaction in such a blocking effect. Our results demonstrated that the spatial distribution of key amino acid residues matters most in the recognition of this toxin towards its channel target rather than its type of fold.
Proteins Structure Function and Bioinformatics 09/2008; 72(2):673-83. · 3.39 Impact Factor
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ABSTRACT: The heat-shock protein Hsp33 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. A crystal was obtained using the hanging-drop vapour-diffusion method and a data set was collected to 2.7 A resolution. The crystal belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 96.43, c = 132.22 A, alpha = beta = gamma = 90 degrees . The asymmetric unit is assumed to contain two subunits of Hsp33, with a V(M) value of 2.96 A(3) Da(-1) and a solvent content of 58.41%.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 03/2007; 63(Pt 2):114-6. · 0.51 Impact Factor
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ABSTRACT: A novel cardiotoxin-like basic protein was isolated from the venom of the Chinese cobra (Naja naja atra) from the south of Anhui in China. The protein inhibits the expression of vascular endothelial growth factor and basic fibroblast growth factor in human lung cancer cell line H1299 and induces the haemolysis of rabbit erythrocytes under low-lecithin conditions. After a two-step chromatographic purification, the resultant 7 kDa protein was crystallized by the hanging-drop vapour-diffusion method at room temperature. A complete data set was collected to 2.35 A resolution using an in-house X-ray diffraction system. The crystal belongs to space group P4(1)2(1)2, with unit-cell parameters a = b = 43.2, c = 147.9 A. There are two molecules in the crystallographic asymmetric unit.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 03/2007; 63(Pt 2):130-4. · 0.51 Impact Factor
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ABSTRACT: The diapause hormone (DH)-pheromone biosynthesis activating neuropeptide (PBAN) gene encodes five neuropeptides, DH, PBAN, alpha-SGNP, beta-SGNP, and gamma-SGNP (subesophageal ganglion neuropeptide). All share the C-terminal pentapeptide FXPRLamide sequence and are produced in the subesophageal ganglion (SG). Expression of the DH-PBAN gene in the central nervous system of embryonic, larval, pupal, and adult Helicoverpa armigera (Har) was studied using in situ hybridization, whole-mount immunocytochemistry, and competitive ELISA. Both Har-DH-PBAN mRNA and protein are localized in the mandibular, maxillary, and labial cell clusters of the SG and a pair of ventral midline neurons of each thoracic ganglion. The FXPRLamide titers in hemolymph are significantly higher in diapause-destined larvae during the fifth and sixth instar than in similar nondiapause-destined individuals. In contrast, the FXPRLamide titers in diapause-destined pupae are significantly lower than in nondiapause-destined pupae. The results from immunocytochemistry and in situ hybridization are consistent with changes of FXPRLamide titers as measured by ELISA. These data suggest that the expression of DH-PBAN might be correlated with diapause induction at the larval stage of diapause-destined individuals and continuous development at pupal stage of nondiapause-destined individuals. Thus, the DH-PBAN gene may play an important regulatory role in aspects of insect development besides diapause termination and pheromone biosynthesis. The transport pathways of FXPRLamide neuropeptides suggest that humoral route is involved in their regulation of development.
General and Comparative Endocrinology 04/2005; 141(1):48-57. · 3.27 Impact Factor
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ABSTRACT: In order to enhance the thermostability of D-glucose isomerase (GI), Gly 138 was decided to be the target to be replaced by molecular design. The mutant G138P was obtained by in vitro site-directed mutagenesis of GI gene. The recombinant plasmid pTKD-GI containing mutant site was expressed in E. coli K38 strain. The comparison experiments of GIG138P with wild-type GI showed that: (1) The half time of GIG138P was as about two times as that of the wild type. (2) The optimum temperature of GIG138P was increased by 10-12 degrees. (3) The specific-activity of GIG138P was similar to the wild-type GI. We supposed, based on the above facts, that the substitution of Pro for Gly at position 138 introduced a pyrrolidine ring, which could just fill perfectly the empty hole leaved by Gly-138 which has no side chain and could make the protein structure more rigid, therefore the mutant G138P enhanced the thermostability of SM33GI.
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 02/1998; 30(6):607-610.
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ABSTRACT: The solution conformations of three hemorrhagic toxins, designated as AaH I, AaH III and AaH IV, from South Anhui Dienagkistrodon acutus have been studied by CD spectra. The secondary structure of AaH I consisted of 25.8% alpha-helix, 12.7% beta-sheet and 26.8% beta-turns, together with 34.7% random coil. For AaH III, the secondary structure contents were 23.9%, 20.6%, 23.7% and 31.8%, and for AaH IV they were 18.2%, 31.0%, 17.2% and 33.8%, respectively. When pH was lower than 4.0 or higher than 11.0, the alpha-helix decreased but beta-sheet increased, meanwhile, the caseinolytic activities of the three toxins decreased. The activities could be inhibited by EDTA, which indicated that all the three toxins were all metalloproteinases. EDTA, Cu(2+), Zn(2+), Ca(2+) and Mg(2+) could change the secondary structures and play an important role in caseinolytic activities.
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 02/1997; 29(2):163-169.
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ABSTRACT: In order to enhance the thermostability of D-Glucose isomerase (GI), its mutants, GIK253R and GIN184V, were obtained with the double primer method of site-directed mutagenesis. Then their biochemical properties were assayed and compared with wild-type GI. The results showed that: (1) The mutant K253R was less stable than the wild-type GI at 70 degrees and 80 degrees. But in 1 M L-Rhaminose at 70 degrees, they had similar rate of heat inactivation. Furthermore, the mutant K253R has higher specific activity than the wild-type GI. (2) The mutant N184V had much less thermostability and specific activity as compared with the wild-type GI. These results were explained by their kinetic parameters and crystal structure model.
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 02/1996; 28(3):272-278.
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ABSTRACT: Thermostability can be increased by introducing prolines at suitable sites in target proteins. Two single (G138P, G247D) mutants and one double (G138P/G247D) mutant of xylose isomerase from Streptomyces diastaticus No.7, strain M1033 have been constructed by site-directed mutagenesis. With respect to the wild-type enzyme, G138P showed about a 100% increase in thermostability, and G247D showed an increased catalytic activity. Significantly, the double mutant, G138P/G247D displayed even higher activity than G247D and better heat stability than G138P. Its half life was about 2.5-fold greater than the wild-type enzyme, using xylose as a substrate. Molecular modelling suggested that the introduction of a proline residue in the turn of a random coil may cause the surrounding conformation to be tightened by reducing the backbone flexibility. The change in thermostability can, therefore, be explained based on changes in the molecular rigidity. Furthermore, the improvements in the properties of the double mutant indicated that the advantages of two single mutants can be combined effectively.
