[Risk factors of pulmonary arterial hypertension in patients with atrial septal defect at high altitude area].

OBJECTIVE: To analyze risk factors of pulmonary arterial hypertension (PAH) in patients with atrial septal defect (ASD) patients living at above 2000 m high altitude area. METHODS: We retrospectively analyzed the incidence of PAH in 526 ASD patients out of 1178 congenital heart disease patients who were hospitalized in Qinghai cardiovascular hospital between January 2007 to December 2009 and explored the risk factors including gander, age, altitude, defect size and nationalities for developing PAH in ASD patients using binary logistic regression. RESULTS: PAH prevalence was 68.4% (360/526) in ASD patients. The risk factors of developing PAH in these ASD patients were defect size (OR: 1.200, 95%CI: 1.156 - 1.246, P = 0.000), age (OR: 1.027, 95%CI: 1.003 - 1.052, P = 0.025) and altitude (OR: 1.389, 95%CI: 1.001 - 1.637, P = 0.043) while gender and nationality were not risk factors for PAH. The incidence of developing PAH increased with aging (P = 0.000). The standardized ratio of PAH at ≥ 3500 m was 74.8% which was significantly higher than that at 2000 - 2499 m altitude (66.2%, P = 0.005) and at 2500 - 3499 m altitude (66.9%, P = 0.005). CONCLUSIONS: The risk for developing PAH is high in patients living at high altitude area. The risk factors of developing PAH in ASD patients living at high altitude are defect size, age and altitude.

[Susceptibility of patients with congenital heart disease to pulmonary hypertension at a high altitude].

OBJECTIVE: To explore the incidence of congenital heart disease (CHD) complicated with pulmonary arterial hypertension (PAH) at an altitude of over 2000 meters above sea level. METHODS: We retrospectively analyzed the profiles of 688 PAH patients selected from 1178 CHD patients at our hospital during January 2007 to December 2009. The risk factors of CHD-PAH were analyzed by age, altitude, etiologies and nationalities. RESULTS: Among 1178 CHD patients, 688 (58.4%) suffered PAH. And 239 PAH cases (54.8%) were found in 436 male patients and 449 PAH cases (60.5%) in 742 female patients (χ(2) = 3.667, P = 0.058). The difference was not significant. The incidences of PAH in CHD patients varied at different altitudes (χ(2) = 10.507, P = 0.005). And 55.2%, 57.2% and 68.7% were found in < 2500 m, 2500 - 3500 m and > 3500 m groups respectively. And the > 3500 m group was statistically higher than those of the < 2500 m and 2500 - 3500 m groups (χ(2) = 9.924, P = 0.005) (χ(2) = 8.007, P = 0.005). The mean pulmonary arterial systolic pressure (PASP) of < 2500 m group was (58 ± 18) mm Hg, 2500 - 3500 m was (63 ± 17) mm Hg and > 3500 m was (64 ± 19) mm Hg. There were significant differences among them. In age composition of CHD-PAH, the highest ratio appeared before the age of 18 years old for PDA (patent ductus arteriosus)-PAH and VSD (ventricular septal defect)-PAH while that of ASD-PAH at the ages of 30 - 39 years old. There were not statistical differences among all nationalities (χ(2) = 5.404, P = 0.248). CONCLUSION: The incidence of CHD-PAH at a high altitude is significantly higher than those at a low altitude. Hypoxia may promote the occurrence and development of PAH in CHD patients.

[A cross-sectional study of congenital heart disease among children aged from 4 to 18 years at different altitudes in Qinghai province, China].

OBJECTIVE: The epidemiological characteristics of congenital heart disease (CHD) in children aged from 4 to 18 years were investigated in Qinghai province. METHODS: Altogether 288 066 children inhabiting at 6 prefectures and 3 counties were examined by the following three steps: pre-screening, re-examination and diagnosis with color Doppler. And the entity distribution was analyzed while the differences were compared by age, gender, altitudes and nationalities respectively. RESULTS: Altogether 1633 cases of CHD were discovered. The total prevalence of CHD was 5.71 per thousand. The prevalence of CHD was found to increase with the increase of altitude by 4.89 per thousand at the altitude of 2535 m, 5.71 per thousand at 3600 m, and 8.74 per thousand at 4200 m respectively. There were significant differences among different altitude (chi(2) = 54.696, P < 0.001). chi(2) trend analysis showed the increase with chi(2) = 41.826(P < 0.001). The total incidence of CHD in females was 6.95 per thousand, which was significantly higher than that in males with 4.54 per thousand (chi(2) = 73.79, P < 0.001). There were significant differences between males and females at the altitude of 3000 m (chi(2) = 84.733, P < 0.001) and 4000 m (chi(2) = 16.313, P < 0.001) except at the altitude of 2000 m (chi(2) = 0.807, P > 0.05). The prevalence of CHD in different age groups was statistically significant at the every altitude of 2000 m (chi(2) = 18.138, P < 0.001), 3000 m (chi(2) = 18.544, P < 0.001) and 4000 m (chi(2) = 27.535 P < 0.001). The prevalence of CHD was increasing with the increase of age groups at the altitude of 3000 m (chi(2) = 19.230, P < 0.001) and 4000 m (chi(2) = 26.894, P < 0.001) except at the altitude of 2000 m. Within the prevalence of CHD of different nationalities, there was a significant difference with chi(2) = 24.456 (P < 0.001). Within the constituent rate of CHD, the prevalence of atrial septal defect (ASD) was as high as 37.42%, followed by the prevalence of patent ductus arteriosus (PDA) as 28.47% and ventricular septal defect (VSD) as 26.01%. Regarding the four categories of CHD, the constituent rate varied at different altitudes. For example, the prevalence rate of ASD constituted 37% at the altitude of 2000 m and 3000 m, and that of PDA accounted for 46.36% at the altitude of 4200 m. CONCLUSION: The epidemiological characteristics of CHD in Qinghai children were possibly associated with altitude levels.

[Antiviral activity of a hammerhead ribozyme against HBV in HepG2.2.15 cells].

The carboxy terminal arginine rich domain of core protein is highly conserved among HBV subtypes, and plays important roles in the packaging of pregenomic RNA and viral replication. Therefore, the 3' highly conserved region of HBV C gene is an attractive target for antiviral therapy mediated by ribozymes. A hammerhead ribozyme RzC, targeting the above site, was designed; meanwhile, as controls, the disabled form of RzC, dRzC, was also designed. In order to investigate its antiviral effects in cultured cells, in vitro-transcribed ribozymeRzC, dRzC and ribozyme expression vectors pCRzC, pCdRzC were delivered, respectively, into HepG2.2.15 cells ( clonal cells derived from HepG2 cells that contain integrated HBV ayw DNA). The preliminary results demonstrated that in vitro-transcribed ribozyme RzC had weak inhibition on HBV replication, possibly due to its quick degradation by RNases in cells, while the endogenously expressed ribozyme RzC showed significant inhibitory effect on HBV replication. In conclusion, the results suggested the possibility of the hammerhead ribozyme RzC to be used for the gene therapy of HBV infection.

Hammerhead Ribozymes Suppress HBV(adr) in HepG2 Cells.

Three hammerhead ribozymes (RS3, RC2 and RC1) targeting to the HBV genome have been designed. Plasmids were constructed by inserting the genes of naked and tRNA-embedded ribozymes into RNA trimming vector pRG523 and then were transferred to eukaryotic expression vector. By the similar cloning method the shotgun-type plasmids carrying homogeneous RS3 or RtS3 unitconnected in tandem were obtained. After co-transfecting the above plasmids and HBV genome containing plasmid into human hepatoma cell line HepG2 respectively and selection by G418, the HBV-inhibiting activity of different kinds of ribozyme in G418-resistant cells was achieved by measuring the decrease of HBV-RNA, progeny DNA and the antigens expressed. The results showed that all the ribozymes were active with more than 70% inhibition activity against the HBV and that tRNA-embedded ribozymes had higher activity than naked ribozymes. It is worth particular interest that shotgun-type ribozymes with the connected unit in tandem with 8 and 12 units constructed in the plasmid revealed the highest activity, reaching >90% inhibition.

Transgenic Silkworm of Anti-NPV Ribozyme.

The plasmid pGL2Rz including ribozyme gene was linearized and introduced into early eggs of silkworm (G(0)) by gene gun. The luciferase activity in blood of the G(1) generation was detected, then the resistant silkworm was selected by NPV infection from G(2) generation. The transgenic silkworm resistant against NPV 10 times more than control ones was got at the G(4) generation. PCR and Southern blotting proved that the ribozyme gene was integrated into the genome of silkworm multicopily. The expression of ribozyme was also detected by RT-PCR in pupa. The results showed that the transgenic silkworm strain of anti-NPV ribozyme has been got.

tRNA-embedded Hammerhead Ribozymes Mediate Destruction of HBV (Subtype adr) in vitro.

Two hammerhead ribozymes, RS3 and RC2, targeting the HBsAg and HBcAg regions of HBV (adr) RNA respectively, have been designed. To improve the stability of ribozymes, two tRNA-embedded ribozymes, RtS3 and RtC2, were obtained by inserting RS3 and RC2 into the anticodon region of tRNA. Although the catalytic activity of the tRNA-embedded ribozymes was lower than that of their naked counterparts, the stability of the former in fetal bovine serum or cytoplasmic extract of HepG2 cells was much higher than that of the naked ribozymes. These results suggest that the antivirus effect of tRNA-embedded ribozymes may be greater in vivo in comparison with naked ribozymes.

Expression of Anti-PCNA mRNA Ribozyme by T7 Vaccinia Virus System in HeLa Cells.

We have designed and synthesized a hammer-head Rz targeted to human proliferating cell nuclear antigen (PCNA) mRNA at site No.524. Results showed that Rz524 can cleave the transcribed substrate RNA site-specifically in vitro. A self trimming expression plasmid of this Rz was constructed and co-introduced into HeLa cells with T7 vaccinia virus. Dot-blotting hybridization showed that the Rz was expressed in HeLa cells.

Purification and Characterization of DNA Helicase BstH1 from Bacillus stearothermophilus.

We have partially purified a DNA helicase BstH1 from Bacillus stearothermophilus through Polymin P precipitation, ammonia sulfate precipitation and column chromatographic steps with Pheny1-Sepharose, DEAE-cellulose, phosphocellulose, FPLC Mono Q and Superose 12. Bsth1 possesses a DNA-Dependent ATPase activity in the presence of Mg(2+). The ATPase activity of BstH1 is differentially stimulated by the presence of different types of nucleic acids. BstH1 has an optimal ATPase activity at 55 degrees. The DNA helicase activity of BstH1 requires a 3'-terminal single-stranded DNA binding site to initiate the unwinding reaction in the 3' right curved arrow 5'direction. BstH1 can unwind blunt-ended duplex DNA in a concentration-dependent manner.

Computer Analysis of the Cleavage Reaction of Hammerhead Ribozyme.

We have analyzed various in vitro cleavage reactions of hammerhead ribozyme by a computer program. Three energy changes: deltaE(s'), deltaE(r'), and deltaE, were calculated. deltaE(s') was the hypothetical energy requirement for the substrate to be activated from the most stable state to the relaxed state. deltaE(r') was the same energy requirement for the ribozyme. deltaE was the calculated free energy decrease by the formation of Rz-S complex from the relaxed ribozyme and the substrate. We found that high deltaE(s') or deltaE(r') reduced the cleavage efficiency of the reaction, and that the deltaE was related to both cleavage efficiency and the optimal temperature of the reaction. deltaE(s'), deltaE(r') can be calculated in advance according to the sequences of the ribozyme and the substrate. Our model, therefore, can be use in the designing of ribozymes.