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A pET system encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH) was introduced into Escherichia coli BL21 (DE3). Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasm of the E. coli transformant. To recover Trx-hPTH from the E. coli culture efficiently, a novel tactic was developed by adding Triton X-100 into the fermentation culture at the exponential growth phase of E. coli and by heat treatment of the culture at the end of the fermentation. A concentration of 1% (v/v) Triton X-100 was added into the culture at the same time as IPTG addition after optimization. Under these conditions, addition of Triton X-100 had little effect on the cell growth, but more than 75% of the total recombinant Trx-hPTH was released into the fermentation broth. Also, a much higher volumetric yield of recombinant Trx-hPTH could be obtained with protein release compared to yield without protein release. Simultaneously, owing to the highly thermal stability of Trx-hPTH fusion protein, heat treatment of the fermentation broth at 80 degrees C for 15 min at the end of fermentation was employed for primary purification. Results demonstrated that heat treatment not only boosted further release of the recombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics described herein integrated the fermentation process with subsequent recovery steps and thus provided a valuable and economical method for the production of Trx-hPTH and maybe some other Trx fusions in E. coli.

Objective: To study the effects of aging and hypoxia on the proliferative behavior of cultured pulmonary arterial smooth muscle cells (PASMCs)., Methods: PASMCs isolated from aged (18-24 months) and young (3-4 months) rats were divided, according to the different treatments the cells were subjected to, into young and aged normoxic groups (groups A and B) and young and aged hypoxic groups (groups C and D) respectively. MTT cell proliferation assay, 3H-TdR incorporation assay, flow cytometriy and immunocytochemical analysis were respectively employed to observe the proliferative behavior., Results: Compared with the cells from young rats, PASMCs from aged rats had a higher proliferation rate, more 3H-TdR incorporation, increased mitotic cell ratio, reduced amount of the total protein, and elevated content of proliferating cell nuclear antigen (PCNA). In comparison with normoxic condition, hypoxia elicited higher proliferation rate of the cells with inhibition of 3H-TdR incorporation that was subsequently increased. Higher percentage of mitotic cells, less total protein amount and increased PCNA were also observed in response to hypoxia., Conclusions: Aging and hypoxia may directly induce PASMC proliferation, and in aging PASMCs, the proliferation is the most obvious in response to hypoxic stimulation.

Objective: To investigate the relationship between endothelial NO synthase (eNOS) gene expression and experimental vasospasm., Methods: Sixteen rabbits were randomly divided into 2 groups, which were subjected to balloon endothelial denudation with normal diet (n=8) or with hypercholesterol diet group (n=8). Angiography was performed to detect the vasospasm induced by ergonovine before and after denudation and 8 weeks after hypercholesterol feeding. In situ hybridization and Northern blotting were performed to localize and quantitate respectively the expression of eNOS mRNA., Results: Visible vasospasm was induced at the denuded sites in rabbits with hypercholesterol diet, in which the expression of eNOS mRNA was detected in the endothelium by in situ hybridization at a lower level than that of rabbits with normal diet, as demonstrated by Northern blotting., Conclusion: The decrement of eNOS mRNA expression resulted from balloon endothelial denudation and hypercholesterolemia may play an important role in the pathogenesis of experimental vasospasm.

OBJECTIVE: To evaluate the long-term effect of percutaneous balloon mitral valvuloplasty (PBMV) by observing the changes in mitral valve reserve after PBMV and during the long-term follow-up and by exploring the relations of these changes with stress echocardiographic scores. METHODS: Stress echocardiogaphy was performed in patients receiving PBMV for mitral stenosis both after surgery and during the long-term follow-up study, and Wilkins scores of the patients were obtained pre-operatively. Intravenous isoproterenol was administered before test to increase the heart rate to simulate mild, moderate and heavy exercises, and the indices for cardiac function were recorded. RESULTS: After PBMV, mitral valve reserve and cardiac function reserve were partly restored, and stress echocardiographic score was closely related to long-term cardiac function ( = 0.82). CONCLUSIONS: PBMV can increase mitral valve area and partly resotreit funtional reserve, and stress echocardiographic score is more predictive of the long-term cardiac function than Wilkins score.