Objective By comparing the change of expression of AQP1 in vitro lung preserved by the continuous infusion of a heart‐lung machine ,continuous pressure perfusion and single low temperature ,to explore the best method of vitro lung preserva‐tion .Methods Thirty Mongrel dogs were randomly divided into 3 groups ,and both lungs were completely resected under the condi‐tion of keeping mechanical ventilation .The vitro lungs were preserved by the way of the continuous infusion of a heart‐lung ma‐chine ,continuous pressure perfusion and single low temperature ,and collecting specimens according to the time point .HE staining was used to observe the morphological changes of vitro lung tissue .Immunohistochemistry and Western blot were used to detect the expression of AQP1 in vitro lung .Results HE staining found that as the time went by alveolar structure gradually collapsed ,in‐flammatory cells increased ,alveolar interval also gradually broadened and exudation could be seen in the alveolar cavity ;at the same time point ,organization structure of extracorporeal circulation group changed lighter than pressure perfusion group and low‐temper‐ature preservation group .In each experimental group ,the expression of AQP1 showed a trend of decline;at each time point ,the ex‐pression of AQP1 in extracorporeal circulation group was higher than pressure perfusion group ,and pressure infusion group was higher than that of low‐temperature preservation group .Conclusion The protective effect of the continuous infusion of a heart‐lung machine on vitro lung was better than continuous pressure perfusion and single low temperature .

AIM: To construct engineered E.coli strains which can express nattokinase with fibrinolysis activity using gene engineering technology. METHODS: The pro-nattokinase (pro-NK) gene was amplified by PCR and inserted into expression vector pET3c. The recombined plasmid pENK which expressed the fusion protein of pro-NK and 22 amino acid peptide was then transferred into lysogenic host strains BL21(DE3)pLysS - and BL21(DE3)pLysS +. Both SDS-PAGE and the fibrin plate assay were used to examine the expression and the activity of the target protein. RESULTS: SDS-PAGE assay showed the fused gene encoding 42 kD fusion protein was expressed in both expression strains pENK-(DE3)pLysS - and pENK-(DE3)pLysS +, and the fibrin plate assay indicated that the expression product had fibrinolysis activity. pENK-(DE3)pLysS - exhibited the basal expression of the target gene, while fusion protein was only induced by IPTG in pENK-(DE3)pLysS +. Basal expression of the fused toxic gene in pENK-(DE3)pLysS - led to bacteriolysis and hollow lawns. CONCLUSION: A pro-NK fusion protein with fibrinolysis activity is successfully expressed in E.coli , which lay a foundation for the exploitation of nattokinase.

To improve the expression level of non fusion hbFGF in E coli , the coding sequence of human bFGF gene, which had been cloned from primarily cultured human fibroblast, was mutated according to the principle of lowering the GC content and increasing the codon preference After being ligated into pET 3c and transformed into BL21(DE3), the recombinant induced by IPTG Expression level was up to 30% of the total bacterial protein The result indicated that optimizing of the TIR would promote the expression level of recombinant protein

Fluorescence enhancement reaction of flavoxate hydrochloride (FX) in strong alkali solution was studied, the mechanism of the reaction was investigated, and a novel fluorimetric method for analysis of FX in drug sample was established. FX has no intrinsic fluorescence, but it can slowly produce fluorescence in strong alkali solution. Heating can promote the fluorescence enhancement reaction. In 3D fluorescence spectra of the decomposition product of FX, two fluorescence peaks, located respectively at excitation wavelengths λex/ emission wavelength λem =223/410 nm, and 302/410 nm, were observed. Using quinine sulfate as a reference, fluorescence quantum yield of the decomposition product was measured to be 0.50. The structural characteriza- tion and spectral analysis of the decomposition product reveal that ester bond hydrolysis reaction of FX is firstly occurred during heating process, forming 3-methylflavone-8-carboxylic acid (MFA), then a cleavage reaction of the γ-pyrone ring of MFA occurred, producing α, β-unsaturated ketone. This product includes adjacent hydroxyl benzoic acid group in its molecule, which can form intramolecular hydrogen bond under alkaline condition, so that increase the conjugate degree and enhance the rigidity of the molecule, and thereby cause fluorescence enhancement. Based on this fluorescence enhancement reaction, a fluorimetric method was proposed for the determination of FX. A linear calibration curve covered the concentration range 0.020 3-0.487 µg · mL. The regression equation was I(F) = 23.9 + 5357.3 c, with correlation coefficient r = 0.999 7 (n = 8), detection limit D = 1.1 ng · mL(-1). The method was applied to the analysis of FX tablets, with a spiked recovery rate of 100.2%. The reliability of the method was verified by a UV-spectrophotometric method.
Alkalies ; Calibration ; Chemistry, Pharmaceutical ; Flavoxate ; analogs & derivatives ; chemistry ; Fluorescence ; Limit of Detection ; Reproducibility of Results ; Solutions ; Tablets

AIM: To get high biological activity of recombinant human bone morphogenetic protein -2 (rhBMP-2) expressed in Escherichia coli by the methods of refolding. METHODS: The rhBMP-2,expressed in Escherichia coli, was washed by Triton X- 100 and further purified by DEAE chromatography.The inclusion bodies were resolved in 8 mol/L urea, and were refolded and dimerized in the redox systems (reduced and oxidized glutathione). Finally, a one - step purification procedure based on the heparin affinity chromatography was implemented. The biological activity of purified rhBMP - 2 were tested by induction of the alkaline phosphatase activity in C2C12 cells. RESULTS: The rhBMP - 2 was expressed in Escherichia coli in a non - active aggregated form of inclusion bodies using a temperature - inducible expression system. The high - purified rhBMP - 2 was obtained in the form of inclusion bodies by several purification courses. The rhBMP - 2 was refolded and dimerized in the redox systems (reduced and oxidized glutathione) and a one - step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP - 2 dimers and monomers. The purified rhBMP - 2 dimers showed the higher biological activity than the commercial rhBMP - 2. CONCLUSIONS: The method achieved the refolding of rhBMP - 2 would be applied to the whole TGF - β superfamily because the BMP - 2 belongs to the superfamily. Meanwhile, the inexpensive,high yield rhBMP - 2 is suitable for clinic application.

AIM: To get high biological activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) expressed in Escherichia coli by the methods of refolding. METHODS: The rhBMP-2, expressed in Escherichia coli, was washed by Triton X-100 and further purified by DEAE chromatography. The inclusion bodies were resolved in 8 mol/L urea,and were refolded and dimerized in the redox systems (reduced and oxidized glutathione). Finally, a one-step purification procedure based on the heparin affinity chromatography was implemented. The biological activity of purified rhBMP-2 were tested by induction of the alkaline phosphatase activity in C2C12 cells. RESULTS: The rhBMP-2 was expressed in Escherichia coli in a non-active aggregated form of inclusion bodies using a temperature-inducible expression system. The high-purified rhBMP-2 was obtained in the form of inclusion bodies by several purification courses. The rhBMP-2 was refolded and dimerized in the redox systems (reduced and oxidized glutathione) and a one-step purification procedure based on the heparin affinity chromatography was implemented to isolate the rhBMP-2 dimers and monomers. The purified rhBMP-2 dimers showed the higher biological activity than the commercial rhBMP-2. CONCLUSIONS: The method achieved the refolding of rhBMP-2 would be applied to the whole TGF-? superfamily because the BMP-2 belongs to the superfamily. Meanwhile, the inexpensive, high yield rhBMP-2 is suitable for clinic application.

Chin ; Dentofacial Deformities ; Humans ; Lip ; Malocclusion ; Maxilla

PURPOSE: This study was aimed to evaluate the effect of the deproteinated bovine bone powder (DBBP) coated with calcium phosphate (Ca-P) on osseous regeneration in the calvarial bone defect of rat. MATERIALS AND METHODS: The DBBP (Control group, n=6) and the Ca-P coated DBBP (Experimental group, n=6) were grafted in the critical sized calvarial bone defect (8 mm) of rat weighing 250 g. The animals were sacrificed at 1, 4 week. The biopsy specimens were decalcified with 5% formaldehyde and embedded in paraffin. The rats were sacrificed at 8 week received tetracycline (1 week), calcein blue (4 week), and alizarin red (7 week), and the biopsy specimens were taken. The specimens were embedded in methylmethacrylate and ground to 10 micrometer thin sections were made. All of the specimens were stained with H & E and Masson's trichrome and examined under light microscope. The specimens at 8 week were examined under fluorescent microscope. RESULTS: In the Control group, the grafted DBBP was surrounded with connective tissue, and osteoblasts were observed partially around the grafted particles at 1 week. At 4 week, some osteoid was observed and, new bone formation was observed at the periphery of grafted materials at 8 week, In the Experimental group, some osteoid was seen at the periphery of the grafted Ca-P coated DBBP at 1 week, and osteoblast and newly formed bone were observed around the grafted materials. At 8 week, newly formed bone was observed at the periphery of the grafted materials. CONCLUSION: These results suggest that Ca-P coated DBBP group was more and faster than DBBP group in new bone formation and Ca-P could contribute to enhance bone formation in the critical sized calvarial bone defect of rat.
Animals ; Biopsy ; Calcium ; Connective Tissue ; Formaldehyde ; Methylmethacrylate ; Osteoblasts ; Osteogenesis ; Paraffin ; Rats* ; Regeneration* ; Tetracycline ; Transplants

Chilblain lupus erythematosus(CLE) is a chronic unremitting from of LE seen predomin antly in women, The chilblain lesion occurs commonly on the digits, calves and heels. Chronic facial discoid LE usually appears before the chilblain form. A 23-year-old female had relatively well defined, erythematous discoid plaques on her both cheeks and scattered erythema multiforme-like ring lesions on her right hand and right wrist. There were also chilblain lesions showing multiple, purple colored macules on her knees, lower legs and periungual areas of fingers and toes. The skin lesions developed at November, 1982 and then the skin lesions remitted during the next summer. The skin lesions recurred at December, 1983.
Cheek ; Chilblains* ; Erythema ; Female ; Fingers ; Hand ; Heel ; Humans ; Knee ; Leg ; Skin ; Toes ; Wrist ; Young Adult

Cardiomyopathies ; diagnosis ; Carnitine ; deficiency ; Humans ; Hyperammonemia ; diagnosis ; Infant, Newborn ; Male ; Muscular Diseases ; diagnosis