To counteract the immune system in parasitic hosts, some viruses encode proteins to suppress the RNA interference (RNAi) effect. In this report, we established two RNAi systems to be easily observed with strong and obvious effect. The function of the P19 of tomato bushy stunt virus, which suppresses RNAi in mammal cells, was then studied using these two systems. Short hairpin RNAs targeting green fluorescence protein (pshRNA-GFP) and firefly luciferase (pshRNA-luc) were designed and inserted into a eukaryotic transcriptional vector pTZU6+1, respectively. The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by the dual-luciferase assay system. pshRNA designed in this study down-regulated the target gene specifically and efficiently, with a decrease of expression of both genes of about 70%, respectively. When P19 was introduced into the RNAi systems, the expression of both GFP and the luciferase were mostly recovered compared with the control groups. The RNAi systems of GFP and luciferase were constructed successfully, demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.

Objective: To observe the effects of manganese( Ⅲ ) meso-tetrakis (N, N'-diethylimidazolium-2-yl) porphyrin (MnTDM) in treatment of early Parkinson's disease(PD) mouse model induced by subcutaneous injection of 1-methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine(MPTP) and to discuss its possible mechanism. Methods:Forty male C57BL/6 mice were evenly randomized into 4 groups: MPTP model group(subcutaneous injection of 25 mg/kg MPTP for 3 days), MnTDM+ MPTP group (15 mg/kg MnTDM was subcutaneously injected 1 h before MPTP injection), MnTDM control group, and normal saline group. Performance of animals in the pole and swimming test was observed 3 days after the last injection. Levels of dopamine (DA) and its metabolites(3,4-dihydroxyphenylacetic acid [DOPAC] and homovanillic acid [HVA]) in the striatum of animals were measured by high-performance liquid chromatography with an electrochemical detector(HPLC-ECD). Thiobarbituric acid (TBA) method was used to examine the levels of malondialdehyde(MDA). Results: Acute injection of MPTP could be used for establishment of PD model. The striatal levels of DA, DOPAC and HVA in MPTP group were significantly lower(P＜0.01)and the striatal level of MDA was significantly higher(P＜0.05) than those of the control group. MPTP had no obvious effect on the behavioral performance of the animals in a short term. MnTDM could partly inhibit the above effects of MPTP. Compared with MPTP group, MnTDM+ MPTP group had significantly higher DA, DOPAC, and HVA levels and significantly lower MDA level(all P＜0.05). There was no significant difference in the behavioral indices of animals between the 4 groups. Conclusion:MnTDM can inhibit lipid peroxidation and promote DA production; it has preventive and therapeutic effects on MPTP induced PD.

Objective Toinvestigate the metabolic characteristics of triglyceride (TG) in the plasma of rabbitswith steroid?induced osteonecrosis of the femoral head and the therapeutic effect of Gushiling Capsule. Methods Rabbit model of femoral head necrosis induced by hormone was established. The level of plasma TG was detected by HPLC/MS and the relationship between the changes of plasma TG level and local histomorphology of femoral head was analyzed. Results Compared with the normal control group, significant changes of plasma TG levels were observed in the model group, the traditional Chinese medicine group and the western medicine group at each time point (P<0.01). However, no significant difference was found between the traditional Chinese medicine group and the western medicine group. Compared with the model group, only a small amount of high signal joint imaging and central signal of the femoral head were found by the MRI examination in the traditional Chinese medicine group and the western medicine group. Pathology assay results showed the degree of femoral head osteonecrosis in the Chinese medicine group was milder than that in the Western medicine group. Conclusion Gushiling Capsule and western medicine Simvastatin have the similar effect of reducing plasma TG, which may be one of the mechanisms underlying Gushiling Capsule preventing steroid?induced femoral head necrosis.

Objective To study the gene expression profile in mice kidney with acute paraquat (PQ) poisoning and identify key genes related to renal injury.Methods A total of 20 mice (C57BL/6) were randomly (random number) divided into four groups, namely control group (group A, n =5) and poisoned groups (groups B, C, D, n =5/group).In group A, mice were administrated with distilled water (0.01 mL/g weight) while in groups B, C, D were administered with equivalent volume of PQ solution (diluted from 20％ to 0.05％ with distilled water) dissolved in distilled water via a gastric tube.Mice of group A were sacrificed immediately and mice of groups B and C at 6 h and 24 h after administration of PQ.The gene expression profile changes of kidney tissue were measured by cDNA Arraychip technology.Mice of group D were observed for mortality rate 48 h later.Results The body weights of mice decreased significantly after administration of PQ.The mortality in group D at 48 h after PQ poisoning was 100％.Compared with the control group, totally 1 792 genes with differential expression variations were identified in 6 h group and 24 h group.There were 8 key genes selected through bioinformatics analysis and they were arranged in real-time PCR: Nlrc5 , Serpinb9 , Cd40 , Rnf135 , Dhx58 , Spl 10 , Fcgrl , and Arhgef12.And then, Nlrc5 , Serpinb9 and Rnf135 were under Western blot investigation.The results of PCR and Western blot showed no significant difference to those from bioinformatics genetic analysis.Conclusions The investigation based on genome wide chip in researching related genes of PQ kidney has offered a novel idea in studying pathogenesis of acute PQ intoxication.

This study was aimed to establish a stable animal model of left ventricular hypertrophy (LVH) to provide theoretical and experimental basis for understanding the development of LVH. The abdominal aorta of male Wistar rats (80-100 g) was constricted to a diameter of 0.55 mm between the branches of the celiac and anterior mesenteric arteries. Echocardiography using a linear phased array probe was performed as well as pathological examination and plasma B-type natriuretic peptide (BNP) measurement at 3, 4 and 6 weeks after abdominal aortic constriction (AAC). The results showed that the acute mortality rate (within 24 h) of this modified rat model was 8%. Animals who underwent AAC demonstrated significantly increased interventricular septal (IVS), LV posterior wall (LVPWd), LV mass index (LVMI), cross-sectional area (CSA) of myocytes, and perivascular fibrosis; the ejection fraction (EF), fractional shortening (FS), and cardiac output (CO) were consistently lower at each time point after AAC. Notably, differences in these parameters between AAC group and sham group were significant by 3 weeks and reached peaks at 4th week. Following AAC, the plasma BNP was gradually elevated compared with the sham group at 3rd and 6th week. It was concluded that this modified AAC model can develop LVH, both stably and safely, by week four post-surgery; echocardiography is able to assess changes in chamber dimensions and systolic properties accurately in rats with LVH.

Objective To explore the relationship between social participation and needs for rehabilitation of the disabled in Guangdong Province and to make a proposal for developing the rehabilitation strategies. Meth-ods The data of the Second National Sample Survey of Disabled Persons in Guangdong Province was used in this study. Ranked data analysis was made with the sub-items of the social participation assessment and the main needs of the disabled individuals. Results Significantly statistical differences were revealed with regard to the constitu-ent ratio of needs for rehabilitation services among people with different degrees of difficulties in social participation caused by hearing and visual impairments as well as physical and mental disabilities. No significant difference was found in terms of the constituent ratio of rehabilitation needs among those with difficulties in speech and those with psychiatric diseases. The major rehabilitation needs focused on medical service, assistive apparatus support and functional trainings. Conclusions The rehabilitation needs were different among different categories of disabled persons. Rehabilitation services should be provided accordingly.

OBJECTIVETo study the effect of HCV NS5A protein on HCV IRES-dependent translation in HepG2 cells.

METHODSHepG2 cells were co-transfected with a plasmid vector containing a bicistronic transcript carrying Renilla luciferase and firefly luciferase genes separated by HCV IRES sequences, and an expressing vector producing the NS5A protein. The luciferase activity and the mRNA of the luciferase gene were then detected. The NS5A expression was confirmed by fluorescence microscopy.

RESULTSHCV NS5A protein was detected in the cytoplasm of the HepG2 cells transfected with pcDNA-NS5A, and the luciferase activity was up-regulated in the presence of the HCV NS5A protein while the expression of luciferase mRNA showed no difference.

CONCLUSIONHCV NS5A protein can upregulate the HCV IRES activity and this effect is dose-dependent with NS5A.

Hep G2 Cells ; Hepacivirus ; genetics ; metabolism ; Humans ; Plasmids ; Protein Biosynthesis ; Protein Structure, Secondary ; Ribosomes ; metabolism ; Transfection ; Viral Nonstructural Proteins ; metabolism

Objective To compare the biological characteristics of eyeballs between Zmu-1:DHP and DHP guinea pigs,and to explore the retinal mechanism of myopia in Zmu-1:DHP guinea pigs. Methods To measure the refraction, corneal curvature and axial length of the two guinea pig strains at age of 4-12 weeks. Those spontaneous myopic Zmu-1:DHP guinea pigs were chosen to take the retina for pathological examination. The pathological changes in the retina were determined and compared with the DHP guinea pigs. The expression of RALDH, ALDHTH, TH, TK, iNOS, nNOS, bFGF and TGFβ mRNA in the retina were detected by real time-PCR. Results The myopic rate of 3-week old Zmu-1:DHP guinea pigs was 90.21%,while of the DHP guinea pig was only 18.00%. From 4 to 12 weeks, compared with the DHP guinea pigs,myopia and axial length of the Zmu-1:DHP guinea pigs were significantly increased(P<0.01),and the corneal curvature of Zmu-1:DHP guinea pigs was significantly less than the DHP guinea pigs(P<0.01). The retina outer nuclear layer of Zmu-1:DHP guinea pigs was reduced in thickness,the cell volume was smaller,and the cell number was less compared with the DHP guinea pigs. The choroid of Zmu-1:DHP guinea pigs showed atrophy and became thinner. There were few pigment granules in the pigment epithelium of Zmu-1:DHP guinea pigs,while there were plenty of pigment granules in the DHP guinea pigs. Compared with the DHP guinea pigs,the expression of TH mRNA was significantly down-regulated in the retina of Zmu-1:DHP guinea pigs(P<0.01),and the expression of TK,iNOS,nNOS,bFGF and TGFβ was significantly down-regulated(P<0.01, P <0.05, P <0.05, P <0.05, P <0.05). Conclusions Zmu-1:DHP strain guinea pig has a high rate of spontaneous axial myopia. The retinal mechanism of myopia has a relationship with the regulation of several myopia factors.

OBJECTIVETo investigate the possible relationship between variation of coxsackievirus B3 (CoxB3) VP1 sequence from cerebrospinal fluid of children with severe and mild central nervous system (CNS) infection and damage to CNS in children from Shandong province.

METHODSThe enteroviruses were detected using VP1 typing and sequencing primer for enteroviruses from 73 enterovirus-infected cases confirmed by detection of cerebrospinal fluid by enteroviruses common primer. VP1 sequences (450 nucleotides) were determined and analyzed for 21 CoxB3 enteroviruses strains isolated in Qingdao and Binzhou, and were compared with that of BLAST search procedures from GeneBank in NCBI. The variation of VP1 gene and amino acids sequence of CoxB3 enteroviruses was analyzed for severe and mild CNS infection.

RESULTSThe nucleotide homogeneity of these CoxB3 appeared to be 97% - 99%, however, the homogeneity among different genotypes were 83% - 76%. Replacement of glutamine by histidine at amino acid locus 856 of VP1 CoxB3 was found in 4 cases with severe encephalitis. There were different variation in VP1 nucleotide sequence of CoxB3 in 3 cases with mild encephalitis and 14 cases with meningitis, but amino acids sequences had no regular variation. The modified Glasgow's coma score was below 7 in all the 4 cases with severe encephalitis. Of these 4 cases, 3 had consciousness disturbance for less than 3 days. Lethargy, restlessness and psychiatric symptoms were major manifestations, of whom 3 also had dysphagia, 1 had encephalatrophy obviously, Glasgow's coma score was 3, deep coma lasted for 9 days, and had concomitant fatal epileptic attacks. Of these 4 cases, 2 completely recovered, 1 had high muscle tone, 1 remained under anti-epileptic drug treatment at follow-up 6 months later.

CONCLUSIONThere were a small epidemic of CoxB3 CNS infection in children in 2005 in this area. The amino acid variation of CoxB3 VP1 possibly caused increased viral virulence and caused damage to CNS.

Amino Acid Sequence ; Base Sequence ; Capsid Proteins ; cerebrospinal fluid ; genetics ; Central Nervous System ; pathology ; virology ; Child ; Coxsackievirus Infections ; cerebrospinal fluid ; epidemiology ; virology ; Encephalitis ; virology ; Enterovirus B, Human ; genetics ; pathogenicity ; Female ; Humans ; Male ; Molecular Sequence Data ; RNA, Viral ; genetics ; Virulence

Objective To explore the relationship between polymorphism of 5-hydroxy tryptamine 2A(5-HT2A)receptor T102C gene and post-stroke depression(PSD),and investigate the heredity-related pathogenesis of PSD.Methods The polymerase chain reaction and restriction fragment length polymorphism(PCR-RFLP)method was applied to test the allele frequencies and genotype of 5-HT2A receptor T102C gene in 97 patients with PSD and 72 patients without depression after stroke.Results The frequency of mutant C allele in patients with PSD was significantly lower than that in patients without depression(P＜0.05),and the frequency of homozygote C/C genotype in patients with PSD was also obviously lower than that in patients without depression(P＜0.05).Conclusion The 5-HT2A receptor T102C gene may be the susceptibility gene of PSD and C allele may be the protective factor of subjects suffering from PSD.