AIM: To improve the acute cumulative death rates method (ACD method) in animal by oral administration. METHODS: A mathematic model was established to evaluate the dose-toxic effect relationship by twice oral administration and the experimental steps were improved too. The methodological quality was explored using the simulation data from computer program and the real experimental date from the reference paper. RESULTS: The results showed that the experimental data could be fitted to its theoretical data from LD_5/2 to LD_ 95/2. Concentration-time curve after po ordinary powder of Semen Strychni in mice were fitted to a one-compartment with T_ 1/2(ka)= 1.136 h,T_ 1/2(ke)= 7.100 h,and T_ max= 3.576 h. CONCLUSION: The improved ACD method can be used in the pharmacokinetics of TCM by oral administration.

Objective To investigate the chemical constituents of Balanophora simaoensis. Methods Chromatography and spectra were used to isolate the constituents and elucidate their structures. Results Four compounds were isolated from whole plant of B. simaoensis and elucidated as methylconiferin (Ⅰ), butylconiferin (Ⅱ), 4-O-?-D-glucopyranosyl confieryl aldehyde (Ⅲ), and brevifolin (Ⅳ), respectively. Conclusion Butylconiferin is a new compound.

Objective To study the relation between the HPV6/18 virus infection and the development of pathological changes of cervix. Methods The number of HPV16/18 DNA copies and the expression rate of HPV16/18 E7 mRNA in the pathological cervix were examined by the quantitative fluorescent PCR combined with pathological diagnosis and immunohistochemistry staining. Results The HPV16 infection rates in chronic cervicitis group were much lower (7.4%) than that in the cervical intraepithelial neoplasia (CIN) groups and the cervical cancer group (69.6% and 72.7%), respectively. Statistical analysis showed that the difference of HPV16 DNA copies was not significant between the chronic cervicitis group and CIN groups. In contrast to the above mentioned result, the number of HPV DNA copies between the CIN groups and the cervical cancer group was significantly different. The HPV16 E7 gene expression rates in CIN Ⅰ, Ⅱ, Ⅲ and cervical cancer groups were 0,37.5%,42.9%,63.6%, respectively. Conclusion Ins more common than that with HPV18. The number of HPV16 DNA copies in cervical cancer tissues is markedly higher than that in CIN Ⅱ, Ⅲ groups. The HPV16 E7 mRNA expression is significantly increased in the cervical cancer, and it is more closely correlated to this pathological changes. The quantitative fluorescent PCR can be used to reflect the activity of HPV, and it is a useful method for the screening examination of HPV and for the early diagnosis and treatment of cervical caner.

Hypertension is classified to fire-heat constitution,yang deficiency constitution,phlegm-wet constitution and yin deficiency causing predominant yang constitution on base of consititutional theory. The therapeutic method of TCM is to modify the unbalance of constitution of hypertension patient.

BACKGROUND:Recent studies have found that long non-coding RNAs (lncRNAs) can regulate stem cel proliferation and differentiation. But it is unclear that how lncRNA NR_033474 regulate stem cel proliferation and cel cycle. OBJECTIVE:To investigate the effect of lncRNA NR_033474 on the proliferation and cel cycle regulation in C3H10T1/2 mesenchymal stem cel s after the NR_033474 overexpressed by lentivirus, and to study the possible regulation mechanism of NR_033474 on mesenchymal stem cel s. METHODS:LncRNA NR_033474 was cloned into a lentivirus vector. Lentivirus particles were infected into C3H10T1/2 cel s to upregulate the expression of NR_033474. The NR_033474 expression level was detected by real-time PCR. Compared with the empty lentivirus vector, the proliferation of C3H10T1/2 cel s which overexpressed NR_033474 was detected by cel counting assay and cel cycle was detected using flow cytometry. The expression of cel cycle-associated proteins such as CDK1, Cyclin B1, Cyclin D1 and P53 were detected by western blot assay. RESULTS AND CONCLUSION:Compared with the control group, lncRNA NR_033474 in C3H10T1/2 cel s which overexpressed NR_033474 was increased by about 100 times (P<0.01), and the proliferation of C3H10T1/2 cel s was significantly inhibited after NR_033474 overexpression by lentivirus (P<0.05). In addition, flow cytometry showed that C3H10T1/2 cel s overexpressing NR_033474 were arrested in G2/M phase compared to the control group. Western blot showed that the expression levels of CDK1 and Cyclin B1 were downregulated, while there were no changes in Cyclin D1 and P53 expression. To conclude, these findings suggest that the NR_033474 overexpression significantly inhibits the cel growth of C3H10T1/2 cel s, at least in part, through induction of cel cycle arrest at G2/M phase.

Objective: To study the distribution of flavonoids among the plants for medicinal use of Selaginella . Methods: HPCE analysis was carried out on the ethyl acetate extracts of 18 Selaginella plants (from 11 species), using MECC separation model. Results: HPCE fingerprints of 18 Selaginella plants were established with quercetin as interanl standard and relative migration time and relative peak area as parameters. Conclusion: The contributions of flavonoids has not only generity, but also difference among the tested samples.

BACKGROUND:Recent studies have found that stem cellpluripotency and differentiation is regulated by many long non-coding RNAs (LncRNAs). The expression and effect of LncRNA AK089560 during differentiation of stem cells is unclear. OBJECTIVE:To investigate the expression of LncRNA AK089560 in mesenchymal stem cells C3H10T1/2 undergoing osteogenic and adipogenic differentiation. METHODS:Osteogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by recombinant human bone morphogenetic protein-2 and evaluated using alkaline phosphatase staining. The adipogenic differentiation of mesenchymal stem cells C3H10T1/2 was induced by three factors (dexamethasone, indomethacin and insulin) and evaluated by oil red O staining. The dynamical expression of LncRNA AK089560 was detected by qRT-PCR assay. The AK089560 secondary structure was predicted using RNAfold software. The relationship between AK089560 and neighboring protein-coding genes was analyzed using UCSC genome browser and visualized by fancyGENE online software. RESULTS AND CONCLUSION:Over 70%of C3H10T1/2 cells were positive for alkaline phosphatase after osteogenic induction and more than 80%of the cells positive for oil red O staining after adipogenic induction. qRT-PCR results showed that the expression of LncRNA AK089560 at days 2, 4, 6 of both osteogenic and adipogenic differentiation was significantly decreased compared with the control group (P<0.05). Bioinformatics analysis showed that there was a stem-loop structure for AK089560 and sense overlap relationship between AK089560 and protein-encoding gene Sema3a. These findings indicate that LncRNA AK089560 expression is reduced during osteogenic differentiation and adipogenic differentiation, showing that AK089560 may be involved in regulating the multi-directional differentiation of stem cells.

4"-O-isovaleryltransferase gene (ist) was regulated by positive regulatory genes of midecamycin 4"-O-propionyltransferase gene (mpt) in Streptomyces lividans TK24. A BamH I ~8.0 kb fragment from Streptomyces mycarofaciens 1748 was proved that it contained mpt gene and linked with two positive regulatory genes, orf27 and orf28. Orf of mpt was replaced by orf of ist and linked with two regulatory genes or orf27 single, and individually cloned into the vectors pKC1139 or pWHM3 (high copy number), and then transformed into S. lividans TK24. The levels of mpt and ist expression were evaluated by the bio-tramsformation efficacy of spiramycin into 4"-O-acylspiramycins in these transformants. The results showed that 4"-O-isovalerylspiramycins could be detected only in the transformants containing the plasmids constructed with pWHM3. The efficacy of bio-transformation of the transformants containing two regulatory genes was higher than that of orf27 single. So, the positive regulatory genes system of mpt gene could enhance ist gene expression.
Acyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Plasmids ; Spiramycin ; analogs & derivatives ; biosynthesis ; Streptomyces ; enzymology ; genetics ; Streptomyces lividans ; metabolism ; Transformation, Genetic

BACKGROUND: Chitosan-disodium β-glycerol phosphate (C/GP) gel has been shown to be compatible with the entrapment of viable chondrocytes, and bone marrow mesenchymal stem cells (BMSCs) are considered to be the potential cells used in tissue engineering. This experiment is aimed to observe the cytocompatibility of BMSCs with C/GP gel.OBJECTIVE: To study the effect of C/GP gel on the growth, proliferation and chondrogenic differentiation in vitro cultured BMSCs and explore a new carrier for the application of cartilage tissue engineering.DESIGN: Completely randomized controlled experiment.SETTING: Department of Bone and Joint Surgery, Affiliated Hospital of Luzhou Medical College; Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA.MATERIALS: The experiment was performed in Center Laboratory of Southwest Hospital Affiliated to the Third Military Medical University of Chinese PLA between October 2005 and April 2006, Six adult female mini-pigss were employed. C/GP gel is prepared by mixture the HCI solution of chitosan with salt solution of β-glycerol phosphate, allowed gel at 37 ℃ in incubator for about 5 to 10 minutes.METHODS: ① BMSCs culture: 4-6 mL of bone marrow harvested from the posterior superior lilac crest were plated at 20 ×10<'6>/100 mm dish and then grown for 14 days in complete media, consisting of DMEM/F-12 supplemented with 10% fetal ovine serum. Cells were harvested and re-seeded for subculture. ② BMSCs differentiation assays: Osteogenic differentiation was assessed by histologic detection of alkaline phosphatase activity and calcium in cultures under osteogenic conditions. Chondrogenic differentiation was evaluated by histology for toluidine blue and immunohistochemistry for type Ⅱ collagen in cultures under chondrogenic conditions. ③ In vitro assays, expanded BMSCs were suspended in C/GP solution and allowed gel at 37 ℃ in incubator for about 5 to 10 minutes, then cultured under chondrogenic conditions for 3 weeks. Cells attached to and viability in C/GP gel was monitored with the aid of an inverted light microscope. Chondrogenic differentiation of cell in C/GP gel were assessed by histological and immunohistocbemistry. The cell proliferated was monitored by MTT after 2, 5, 8 days seeding.MAIN OUTCOME MEASURES: ① Characterization of mini-pigs' BMSCs; ② BMSCs attached to and viability in C/GP gel; ③ Chondrogenic differentiation of BMSCs in C/GP gel; ④ BMSCs proliferated in C/GP gel.RESULTS: ① Characterization of mini-pigs' BMSCs: Cultured BMSCs showed fibroblastic morphology and were able to differentiate to chondrocytes or osteogenic cells under chondrgenic or osteogenic cultured condition respectively. ②BMSCs attached to and viability in C/GP gel: BMSCs attached to and remained > 90% viable in C/GP gels immediately post-casting, and throughout the 21 days, using MTT staining. ③ Chondrogenic differentiation of BMSCs in C/GP gel: During 21 days culture period in vitro, chondrogenic induced BMSCs produced amounts of de novo cartilage matrix in the chitosan, as assessed by histological and biochemical criteria. ④ BMSCs proliferated in C/GP gel: Chondrogenic induced BMSCs cultured in C/GP gels continued to proliferate. There was a significant difference among the values of optical density in the cells-gel constructs compared to the controls without cells after 2, 5, and 8 days of culture (P<0.05).CONCLUSION: It is confirmed that C/GP gel shows good cytocompatibility with BMSCs and contributes to the growth, proliferation and chondrogenic differentiation for BMSCs in vitro culture. C/GP gel can be a potential cell-carrier for tissue engineering of articular cartilage.

A simple and rapid method was developed based on high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to determine sivelestat and its metabolite XW-IMP-A in human plasma. After a simple protein precipitation, the samples and internal standards were analyzed on a C18 column by a gradient elution program. The mobile phase consisted of 30% acetonitrile in methanol and 5 mmol · L(-1) ammonium acetate at a flow rate of 0.7 mL · min(-1). The mass spectrometric data was collected in multiple reaction monitoring mode (MRM) in the negative electrospray ionization. The standard curves were linear in the range of 10.0-15,000 ng · mL(-1) for sivelestat, and 2.50-1000 ng · mL(-1) for XW-IMP-A. The low limits of quantitation were identified at 10.0 and 2.50 ng · mL for sivelestat and XW-IMP-A, respectively. The intra- and inter-day precision were within 11.3% and 13.1% for sivelestat and XW-IMP-A, and accuracy was 0.3% and 0.6% for sivelestat and XW-IMP-A, within the acceptable limits across all concentrations. The method was successfully validated in the pharmacokinetic study of sivelestat in healthy Chinese volunteers.