[Optimization of determination methods of astragaloside â £ and analysis of contents of astragaloside â £ in different origin and grade].

A rapid and accurate method for determination of astragaloside Ⅳ was established,which was further applied to determine the contents of astragaloside Ⅳ in 87 batches of different origin and different grade of Astragali Radix. The ROC curve was used to analyze the contents of astragaloside Ⅳ in different origin. Simultaneous contents of astragaloside Ⅳ in different grade were compared with chemometrics. HPLC-ELSD method was used to determine the contents of astragaloside Ⅳ. A Vensil MP C18 column( 4. 6 mm×250 mm,5 µm) was used with acetonitrile-water( 32 ∶68) as the mobile phase at a flow rateof 1 m L·min-1. The column temperature was 25 ℃ with ELSD parameters as follows: gas flow rate was 2. 5 L·min-1,the drift tube heating temperature was set to 105 ℃,and the gain value was 4. 0. The optimized method avoided the problem that the consumable quality unstable and the recovery rate was not high. The contents determined by the optimized method were higher than the pharmacopoeia method,with less time and high recovery rate. The ROC curve analysis showed that there was no significant difference of contents of astragaloside Ⅳ between the top grade of Shanxi wild-simulated Astragali Radix top and the first grade of Gansu cultivated Astragali Radix. The contents of astragaloside Ⅳ in the second,third and fourth grade of Shanxi wild-simulated Astragali Radix was significantly higher than those of produced from Gansu.There was a significant negative correlation between the contents of astragaloside Ⅳ and grade in Shanxi Astragali Radix. While there was no correlation for Gansu Astragali Radix. This study provided the basis for the quality grade standard of Astragali Radix.

[Studies on mechanism of protective immunity against infection of Schistosoma japonicum induced by Sj26 gene transfected dendritic cell].

OBJECTIVE: To explore the mechanism of protective immunity against Schistosoma japonicum infection induced by Sj26 gene transfected dendritic cell (DC). METHODS: 48 BALB/c mice were divided randomly into 4 groups with 12 each. The mice were injected through auricle for three times with Sj26 gene transfected DC (Group A), pcDNA3 transfected DC (Group B), untreated DC (Group C) and RPMI-1640 (Group D) respectively, and challenged with 40+/-2 cercariae of S. japonicum per mouse 2 weeks after the last immunization. Sera from mice were examined for IgG antibody, IFN-gamma and IL-4 by ELISA. Western blot was used for detecting specific anti-Sj26 IgG antibody. The production of IFN-gamma and IL-4 in the supernatant of spleen cells stimulated with soluble egg antigen (SEA) and ConA was quantified by sandwich ABC-ELISA. The proliferation of spleen cells were measured with MTT method. RESULTS: IgG antibody increased significantly in the mice of group A at 2 weeks after the last immunization (absorbency A491=0.117), higher than that of group B (A491=0.061) and group C (A491=0.058) (P<0.05). The Mr 26000 antigen of S. japonicum was strongly recognized by sera from group A by Western blot. The level of IL-4 in mice of each group showed no significant difference before and after immunization. The level of IFN-gamma in group A (101.4+/-4.9 pg/ml) was significantly higher than that before immunization (15.0+/-1.9 pg/ml) and that of group B (40.1+/-3.1 pg/ml) and group C (35.6+/-1.2 pg/ml) (P<0.01). The level of IFN-gamma in spleen cells from group A in response to ConA and SEA (171.2 and 70.8 pg/ml, respectively) was higher than that of group D (91 and 49.7 pg/ml, respectively) (P<0.01). The level of IL-4 in spleen cells from group A in response to ConA and SEA (79.7 and 50.7 pg/ml, respectively) was lower than that of group D (125.2 and 70.5 pg/ml, respectively) (P<0.01). The stimulating index of spleen cells from group A was 4.1 and 2.82 in response to ConA and SEA respectively, higher than that of other groups (compared with group D, P<0.05). CONCLUSION: Sj26 gene transfected dendritic cell induces predominant Th1 type immune response which might play a role in protection against S. japonicum infection.

Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage.

A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5'-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3'-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn't. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.

[Localization of EGFP-DPF-1 expressed and secreted by HeLa cells in oocytes].

The eukaryotic expression plasmid pEGFP-N1/DPF-1 was constructed through fusing rabbit oviduct-specific glycoprotein (DPF-1) gene to the 5' terminus of eGFP gene. After transfecting the recombinant plasmid into HeLa cells, we got some cell strains expressing and secreting eGFP-DPF-1 stably. The apparent molecular weight of the fusion protein was up to 120 KD indicating the fusion protein was modified. Rabbit oocyte-cumulus cells complexes (COC), the cumulus cells-deprived oocytes or oviductal oocytes were co-cultured with the cell strain or cultured in the conditioned medium derived from the cell strain to observe the distribution of DPF-1 in oocytes after incubation. The results revealed that DPF-1 but not GFP alone associated with zona pellucidae (ZP) of oocytes, especially in the inner layer of ZP, and was evenly distributed in dot-like shape on the outer surface of the cytoplasmic membrane. In contrast, the cumulus cells around the oocyte did not interfere the association of DPF-1 with oocyte, and no trace of DPF-1 was found in the cumulus cells. The experiment proved that the oviductin expressed and secreted by eukaryotic cells in vitro could bind to oocytes, and was directly observed by using green fluorescent protein as a tracer. These results provided some interesting informations for the further studying on the functions of the oviductin.

[Factors of effects on transformation behaviors of inorganic mineral matters in desulfurizing process of burning coal].

The Heshan coal and the Wansheng coal were selected to investigate the effects of Ca/S molar ratio, combustion temperature and other factors on the desulfurization efficiency when limestone as a desulfurizer. And then the possible effects of various desufurizing systems on the mineral constituents of their residues were discussed by using the X-ray diffraction analyzer. Moreover, the phase diagrams of the ternary system CaO-Al2O3-SiO2 were further used in order to find out the transformation behaviors of inorganic mineral matters in the desulfurizing processes of burning coal on the basis of the XRD results. The results show that the combustion temperature and the Ca/S molar ratio are the key factors affected the desulfurization efficiency and their residues' mineral constituents. When the temperature raised and the Ca/S ratio increased, the solid-phase reactions between CaO and some inorganic matters in coal ash will be obviously accelerated, which thus are good for the formaion of many mineral matters such as C4AF,C2AS and beta-C2S. Yet the anhydrite formed will decompose if the temperature is above 1050 degrees C and then the desulfurization efficiency will decrease. The desulfurization efficiency in different temperature is mainly decided by the existence form of the sulfur-contained mineral matters and their contents in the residue. In the experiment, the best desulfurization efficiency for the HS coal was about 90% when the Ca/S ratio was 2 or 3 and the range of the temperature was from 850 degrees C to 950 degrees C.

[Cloned goats produced from the somatic cells of an adult transgenic goat].

This study was carried out to examine the effect of different donor cell type and micro-manipulation on the development of reconstituted embryos. Cultured mural cumulus cells or fibroblast cells from an adult transgenic goat expressing human erythropoietin(rhEPO) were used as the donor cells in nuclear transfer experiments. The reconstituted eggs were generated by transferring fibroblast cells or cumulus cells into the perivitelline space of enucleated M II oocytes and then followed by electrofusion and activation. After 6 days' incubation in vivo, the reconstructed embryos developed into morulae or blastocysts were transferred into 6 foster recipients. Two of the foster-mothers were pregnant and gave birth to two offspring, which were derived from the fibroblast cell and cumulus cell, respectively. Fingerprint analysis showed that the PCR-RFLP patterns of the two offspring were identical to that of donor goats. PCR results indicated that these cloned goats carried hEPO gene as same as their donor cells.