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.
Blotting, Western ; Cell Line ; Cell-Penetrating Peptides ; biosynthesis ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Gene Products, tat ; Genetic Vectors ; Globins ; biosynthesis ; Humans ; Hydrogen Peroxide ; Recombinant Fusion Proteins ; biosynthesis

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.
Animals ; Carbon Tetrachloride ; Chemical and Drug Induced Liver Injury ; prevention & control ; Free Radical Scavengers ; metabolism ; Globins ; biosynthesis ; genetics ; therapeutic use ; Liver ; enzymology ; Male ; Mice ; Polyethylene Glycols ; chemistry ; Protective Agents ; therapeutic use ; Recombinant Proteins ; biosynthesis ; genetics ; therapeutic use

OBJECTIVE: To investigate the effect of extracts with water and alcohol from Radix Trichosanthis on the cell survival and the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg) in HepG2.2.15 cell supernatant. METHODS: The cell survival rate of HepG2.2.15 cells was detected by MTT assay. The HBsAg and HBeAg in HepG 2.2.15 cell supernatant were evaluated by enzyme linked immunosorbent assay. RESULTS: The water and alcohol soluble extracts from Radix Trichosanthis significantly inhibited the levels of HBsAg and HBeAg in HepG2.2.15 cells in a time-and-concentration-dependent manner. However, the therapeutic index of extracts with water from Radix Trichosanthis was better than that in the alcohol group. CONCLUSION The activity of water-soluble extract from Radix Trichosanthis is stronger on anti-hepatitis B virus than that of the alcohol-soluble extract.
Antiviral Agents ; pharmacology ; Drugs, Chinese Herbal ; classification ; pharmacology ; Hep G2 Cells ; Hepatitis B Surface Antigens ; biosynthesis ; Hepatitis B e Antigens ; biosynthesis ; Hepatitis B virus ; drug effects ; Humans

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.
Animals ; Cell Line ; Globins ; genetics ; pharmacology ; Hepatic Stellate Cells ; cytology ; pathology ; Humans ; Hydrogen Peroxide ; toxicity ; Oxidative Stress ; drug effects ; Protective Agents ; pharmacology ; RNA, Small Interfering ; genetics ; Rats ; Reactive Oxygen Species ; metabolism

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.
Capsid ; physiology ; Capsid Proteins ; genetics ; DNA, Viral ; genetics ; Dependovirus ; genetics ; physiology ; Genetic Vectors ; Genome, Viral ; Virus Assembly ; genetics ; physiology

OBJECTIVEThis study aims to investigate the protection of procyanidins and lycopene from the renal damage induced by mercuric chloride.

METHODSRats were treated with either procyanidins or lycopene 2h before HgCl(2) subcutaneously injection, once daily treatment for 2 successive days.

RESULTSIn comparison with HgCl(2) group, markers of renal function such as blood urea nitrogen in serum and urinary protein were decreased to (18.45±11.63) mmol/L and (15.93±9.36) mmol/L, (4.54±0.78) g/(g·Cr) and (4.40±1.12) g/(g·Cr). N-acetyl-beta-D-glucosaminidase, lactate dehydrogenase, alkaline phosphatase in urine were depressed to (125.49±11.68) U/(g·Cr), (103.73±21.79) U/(g·Cr), (101.99±12.28) U/(g·Cr), and (113.19±23.74) U/(g·Cr), (71.14±21.80) U/(g·Cr), (73.64±21.51) U/(g·Cr) in procyanidins and lycopene groups. Indicators of oxidative stress, for example, Glutathion was reduced to (45.58±9.89) μmol/(g·pro) and (45.33±5.90) μmol/(g·pro), and antioxidant enzymes such as superoxide dismutase, glutathione-peroxidase were enhanced to (43.07±10.97) U/(mg·pro) and (39.94±6.04) U/(mg·pro), (83.85±18.48) U/(mg·pro), and (85.62±12.68) U/(mg·pro). Malondialdehyde was lowered to (0.95±0.12) (μmol/g·pro) and (1.03±0.12) μmol/(g·pro) in procyanidins and lycopene groups. ROS generation was decreased by 27.63% and 16.40% and apoptosis was also decreased in procyanidins and lycopene groups respectively. Pathological changes were much better as well.

CONCLUSIONProcyanidins and Lycopene play some protective role against mercury kidney damage.

Acetylglucosaminidase ; urine ; Alkaline Phosphatase ; urine ; Animals ; Antioxidants ; therapeutic use ; Blood Urea Nitrogen ; Carotenoids ; therapeutic use ; Glutathione ; metabolism ; Glutathione Peroxidase ; metabolism ; Kidney ; drug effects ; metabolism ; pathology ; Kidney Diseases ; chemically induced ; metabolism ; pathology ; prevention & control ; L-Lactate Dehydrogenase ; urine ; Lipid Peroxidation ; Malondialdehyde ; metabolism ; Mercuric Chloride ; pharmacokinetics ; toxicity ; urine ; Mercury ; metabolism ; Proanthocyanidins ; therapeutic use ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism

Purpose To investigate human soluble TRAIL(sTRAIL)protein expression and purification and its potential anti-tumor activity on hepatocellular carcinoma(HepG2).Methods Soluble TRAIL gene ligated with expression vector pPIC9 was transfected into GS115(his4)and the recombination strain expressing sTRAIL was screened by MD plate.The effects of different media,methanol inducement period,methanol concentration,and pH were investigated and optimized using shaking flask.The anti-tumor activity of sTRAIL with HepG2 cells Was analyzed after purification.Results The highest expression of sTRAIL was obtained at pH 6.0,1% methanol in BMMY medium,with the concentration of(58.7±2.4)mg/L at 48 h.Recombinant sTRAIL protein could induce HepG2 cells apoptosis and inhibit HepG2 cells proliferation effectively.Conclusion The optimized condition of human sTRAIL expression and purification Was developed and the obtained recombinant sTRAIL protein may be a promising therapeutic agent for hepatocellular carcinoma.

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.
Antioxidants ; pharmacology ; DNA, Complementary ; Electroporation ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Serpins ; biosynthesis ; genetics

Objective:To construct K-ras-targeted siRNAs (K-ras siRNA) and to investigate the inhibitory effects of K-ras siRNAs on the growth and migration of lung cancer A549 cells (containing mutant K-ras gene) and NCI-H446 cells (containing wild-type K-ras gene). Methods: Four K-ras siRNAs (K-ras siRNA1～K-ras siRNA3 targeting wild-type K-ras and K-ras siRNA4 targeting mutant K-ras) were designed and artificially synthesized; they were used to transfect A549 cells and NCI-H446 cells. The expressions of Ras mRNA and protein were examined by RT-PCR and Western blot-ting. The inhibitory effects of K-ras siRNAs on the proliferations of A549 and NCI-H446 cells were determined by MTT assay. The effects of K-ras siRNAs on the cell migration and apoptosis were observed by Transwell assay and Hoechst 33258 staining, respectively. Results: Mutant K-ras-targeted siRNA (K-ras siRNA4) specifically inhibited the K-ras ex-pression but had no influence on H-ras and N-ras expression in A549 cells. K-ras siRNA4 inhibited the proliferation of A549 cells but did not inhibit that of NCI-H446 cells, which contained wild type K-ras gene. K-ras siRNA4 also induced apoptosis and inhibited migration of A549 cells. Conclusion: Mutant K-ras-targeted siRNA4 can inhibit the proliferation, migration and induce apoptosis of A549 cells. It may be a potential and personalized drug for the treatment against lung cancer containing mutant K-ras gene.

AIM To study whether chlorpromazine(CPZ) and verapamil (Ver) have protective effects on the nephrotoxicity of cadmium (Cd). METHODS Thirtytwo Wistar rats were divided randomly into four groups.Each agent was injected 5 times per week for 6 weeks.The rats in Cd-treated group were sc injected with CdCl2 7μmol·kg-1. The rats of CPZ- and Ver- pretreated group were ip injected with CPZ 5 mg· kg- 1, Ver 4 mg· kg- 1,respectively, 1 h later sc injected with CdCl2 7 μ mol·kg- 1. The control group was sc injected with saline 2 mL·kg-1 at corresponding time. Twenty-four hours after the last injection, the 24-h urine samples were collected. The renal cortex was also excised. Lactate dehydrogenase (LDH) activity, protein and Cd concentration in urine were determined.The activities of protein kinase C (PKC), Na + -K + -ATPase, Ca2 + -ATPase and Cd concentration of renal cortex were also measured. RESULTS Cd concentrations of renal cortex and urine in rats from Cd-treated group were significantly higher than those of control group.Cd concentrations in urine of rats from CPZ- and Ver-pretreated groups were significantly lower than those of Cd-treated group, but there was no significant change in renal cortex. As compared with control group, LDH activity, protein content in urine and the activities of PKC, Na+ -K+ -ATPase and Ca2+ -ATPase in the rats of Cd-treated group increased significantly. LDH activity, protein content in urine and activities of PKC,Na+ -K+ -ATPase and Ca2+ -ATPase in rats of CPZ- and Ver-pretreated groups were significantly lower than those of Cd-treated group. CONCLUSIONCd could activate the activities of PKC,Na+-K+-ATPase and Ca2+-ATPase. Moreover, pretreatment of CPZ and Ver could reduce nephrotoxicity of Cd.