OBJECTIVEExpress and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.

METHODSThe expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.

RESULTSSuitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.

CONCLUSIONSWe have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.

DNA, Single-Stranded ; genetics ; metabolism ; DNA-Binding Proteins ; chemistry ; isolation & purification ; metabolism ; Hepacivirus ; Hepatitis C ; metabolism ; virology ; Humans ; Molecular Weight ; Protein Binding ; RNA, Viral ; genetics ; metabolism

AlM: To describe the expression of hypoxia-inducible factor-1α ( HlF-1α) and erythropoietin ( EPO ) in rats' corneal and evaluate its potential effect on corneal neovascularization ( CNV) growth.
METHODS:The young SD rats (3mo) was chosen and randomly divided into 2 groups, which were experimental group and normal control group. CNV model was established by alkali burn, and the length and area of CNV was observed everyday after operation by slit lamp. After that, the expression of HlF-1α and EPO was measured by SABC and RT-PCR methods at 1, 3, 5, 7, and 14d after alkali burn. The data was analyzed by SPSS 20. 0.
RESULTS:The area of CNV was increasing at 1, 3, 5, 7, and 14d after alkali burn, and the peak point appear at 7d. The growth speed was decreased after 14d. SABC method told us that no HlF-1αand very tiny amount EPO was detected at normal rats' corneal. The expression of the two factors increased at 1d after alkali burn in corneal epithelium and endoderm. The results of RT - PCR showed that a few amounts of HlF-1α and EPO mRNA were detected at normal group. The expression of the two factors was increased at 3d after alkali burn, and the peak value was found at 7d, however, it was decreased at 14d. Statistical difference was found at different time (P<0. 05).
CONCLUSlON: HlF- 1α and EPO is closely related to CNV.

Objective To evaluate the efficacy and safety of dorsal ligament reconstruction in treatment of old dorsal dislocation of distal radioulnar joint.Methods Seven patients with old dorsal dislocation of distal radioulnar joint were treated with dorsal ligament reconstruction using the palmaris longus tendon from March 2005 to May 2012 in our institute,including 4 males and 3 females with a mean age of 37 years.All patients had a history of wrist injury for more than 3 months and were diagnosed as isolated dislocation of distal radioulnar joint without fractures.During the operation a bone tunnel was made at dorsal ulnar side of radius near the ulnar notch,which was parallelized to long axial of ulna,two holes were drilled from dorsal to palmarulnaris side through the extensor carpi ulnaris sulcus of the ulna.The palmaris longus tendon was harvested and the strip of the tendon was penetrated through the radial hole.After the tips being crossed,put them through the holes of ulna,reduct the distal radioulnar joint by supinating the forearm,the strip of the tendon was sutured after being tightened,the reversed back the free end of the tendon to reconstruct the sheath of extensor carpi ulnaris tendon.Postoperatively,the upper extremity were kept in a long arm plaster in the position of elbow flexion 90° and forearm supination for 3 weeks,then the below elbow cast was replaced for another 3 weeks.Results Patients were followed-up for 1 year and 8 months 4 years and 2 months with the average of 2 years and 9 months.The rotation of wrist was improved and the handgrip strength was increased significantly.A functional evaluation was performed using the modified Mayo wrist scoring system.All patients had better wrists scores postoperatively (mean,93) compared to preoperatively (mean,68).All patients satisfied with the final result.Conclusion Dorsal ligament reconstruction should be a promise surgical modality for the old dorsal dislocation of distal radioulnar joint.

Objective:To investigate the effects of shikonin on osteoclastogenesis in vitro and amelioration of bone loss in ovariectomized-induced osteoporosis in mouse model.Methods:The optimal concentration of shikonin treating were evaluated in vitro depending on its effect on the viability of C57BL/6J mouse bone-marrow-derived macrophages by CCK-8 method.To establish the osteoclastogenesis cell model,macrophages were cultured with RANKL and M-CSF treatment,and TRAP staining was used to observe the generation of osteoclasts after treating with different concentration of shikonin solution.Expressions of osteoclast marker genes,including TRAP,c-Fos and NFATclwere detected with real-time PCR.Fifthteen mice were randomly allocated into sham operation group,ovariectomized model group and shikonin treatment group.After the modeling,mice in treatment group were received the intraperitoneal injection of shikonin,while the other two groups treated with normal saline.After thirty days treatments,all animals' tibias were dissected for micro-CT analysis.Results:①The macrophages viability was significantly inhibited when the concentration of shikonin was higher than 250 nmol/(P＜0.01).②The osteoclastogenesis was significantly suppressed by differemt dose of shikonin(P＜0.01).③ The expression of the osteoclastic marker genes (TRAP,c-Fos and NFATc 1) were suppressed by addition of shikonin comparing to control group (P＜0.01).④ Shikonin effectively prevented ovariectomy-induced bone loss (P＜0.05).Conclusion:Shikonin suppresses osteoclastogenesis in vitro and ameliorates ovariectomized-induced osteoporosis in mouse model.

AIM: To study the protective effects of Imipramine (Imi) on ischemic injury in cultured rat cerebral cortical neurons. METHODS: Cortical neurons of fetal rat were cultured in vitro. The protective effects of Imi on ischemic injury in cultured rat cerebral neurons were observed. RESULTS: Imi ( 10 -5, 10 -6, and 10 -7 mol?L -1) reduced the number of cell death, lowered LDH,NO,and MDA content, and increased of activity of SOD. CONCLUSION: Imi can protect rat cerebral cortical neurons from ischemic injury and toxicity of Glu. And it maybe attribute its to antioxidation and calcium antagonism.

Objective To explore different doses of calcium 5-formyltetrahydrofolate(CF)for protecting enteral mucosa after chemotherapy of high-dose methotrexate(HD-MTX) in rats.Methods Sixty of 6 weeks old Wistar rats were divided into 5 groups in random,12 rats every group.Group A:control group,normal sodium(NS) intraperitoneal injection only;Group B to E:after HD-MTX intraperitoneal injection(120 mg/kg),1% CF(CF dose amounts to 1% of total MTX dose) for group B,2% CF for group C,8% CF for Group D and empty for group E.For group B、C and D,CF were intramuscular injected after 12 hours of MTX used,q6h?7 times.Rats were killed after 18 hours of the last time of CF.Morphous of jejunum dissection were observed and length of intestinal villus and depth of crypt were mea-sured.Results For group A,jejunum walls were thick and elastic and intestinal villus were close and orderly.Jejunum walls were congestive,swollen and thin,length of intestinal villus and depth of crypt reduced both in group B to E.These were most obvious in group E,and were secondary in group B.Statistical analysis showed that significant difference in effect existed between group B,C,D,E and group A(Pa0.05).Conclusion MTX can damage in intestinal mucosa of rats,CF can reduce this damage,excessive low doses of CF can't play this role.

OBJECTIVE To investigate the mechanism of SIRT1/AMPK signaling pathway between hepatocytes and hepatic stellate cells (HSCs). METHODS Normal human Chang liver cells and human hepatic stellate cell line, LX-2 cells were treated with SRT1720 (10 μmol·L-1) and AICAR (500 μmol·L-1) prior to ethanol (50 mmol·L-1) for 24 and 48 h. Cell viability was analyzed by methyl thiazolyl tetrazolium assay. SIRT1, AMPK and p-AMPK mRNA levels for 24 h and 48 h were analyzed by RT-PCR, SIRT1, AMPK and p-AMPK protein expressions in the supernatant at 24 and 48 h was detected by Western blot. RESULTS SRT1720 and AICAR effectively decreased LX-2 cell viabilities and exhibited scarcely little toxicity in human Chang liver cells. SRT1720 and AICAR attenuated collagen-I, α-smooth muscle actin (α-SMA) levels, activated liver kinase B-1 (LKB1) and AMPK phosphorylation in ethanol treated LX-2 cells. Meanwhile, SRT1720 and AICAR enhanced SIRT1 expression mediated by ethanol both in Chang liver cells and LX-2 cells. Furthermore, SRT1720 and AICAR suppressed the expression of sterol regulatory element-binding protein-1 (SREBP-1) to regulate fatty acid synthesis. CONCLUSION SIRT1 agonist and AMPK agonist blocked the crosstalk between hepatocytes and HSCs via SIRT1/AMPK signaling pathway to modulate hepatocytes accumulation of lipid and HSCs activation.

Objective: To investigate the impact of SARS on heart initially. Methods: the clinical and laboratory data of 86 patients with SARS were collected and analyzed statistically. Results: In recovery phase, the rest heart rates in 64% of patients with SARS exceeded 90bpm, and the heart rates after mild exercises in 72.1% of patients with SARS exceeded 100bpm. Conclusion: SARS, which mainly resulted in pulmonary damage, may involve heart.

OBJECTIVE To investigate the hepato-protective mechanism of thymoquinone (TQ) onthe development of acetaminophen (APAP)- induced liver injury. METHODS In vivo, male kunming mice were injected with a single dose of 300 mg·kg-1 APAP. Some mice were pretreated with TQ (5 or 20 mg·kg-1) and N-acetylcysteine (NAC, 300 mg·kg-1) 2 h before APAP injection. Mice were euthanized at 2 h, 6 h, 12 h after APAP treatment. In vitro, human Chang liver cells were incubated with 3.125, 6.25 or 12.5 μmol·L-1 TQ, 10 μmol·L-1 SP600125 and 500 μmol·L-1 AICAR in the presence of APAP for 24 h. Cell viability were analyzed by MTT assay, protein expressions were assessed by Western blot. RESULTS TQ pretreatment significantly reduced serum aminotransferase and increased hepatic gluta?thione (GSH) and glutathione peroxidase (GSH-PX) activities, while significantly inhibited interleukin-1β(IL-1β) levels. TQ significantly inhibited c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK) and P38 phosphorylation induced by APAP. Moreover, TQ inhibited phosphatidylinositol 3- kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling activation and activated AMPK phosphorylation induced by APAP. In addition, TQ inhibited signal transducer and activator of transcription 3 (STAT3) phosphorylation on APAP-induced liver injury. In vitro, APAP enhanced JNK phosphorylation and attenuated AMPK phosphorylation in Chang liver cells, and these effects were blocked by pretreatment with TQ, SP600125 (JNK inhibitor) and AICAR (AMPK activator). CONCLUSION Our findings suggest that TQ may actively prevent APAP-induced liver injury, and this effect may be mediated by JNK and AMPK signaling pathways.

OBJECTIVETo investigate the effects of antisense oligonucleotide specific to K-ras point mutation on human pancreatic carcinoma cell PC-2 in vitro.

METHODSHuman pancreatic carcinoma cell PC-2 was transducted with antisense oligonucleotide specific to K-ras point mutation by liposome; the expression of target gene was studied with immunohistochemistry and in situ hybridization. The effect on cell proliferation was studied by artificial count, MTT and mass test.

RESULTSThe expression degree of ras protein and K-ras mRNA transducted with antisense oligonucleotide decreased apparently compared with control group and sense oligonucleotide group 48 h after tansduction. The inhibitory effect on cell proliferation was confirmed by artificial count, MTT and mass test.

CONCLUSIONSAntisense oligonucleotide specific to K-ras point mutation has an apparent inhibitory effect on target gene expression and cell proliferation of human pancreatic carcinoma cell in vitro.

Cell Proliferation ; drug effects ; Genes, ras ; genetics ; Humans ; Oligonucleotides, Antisense ; genetics ; pharmacology ; Pancreatic Neoplasms ; genetics ; pathology ; Point Mutation ; genetics ; Transfection ; Tumor Cells, Cultured