The concentration of 5 nucleosides, uracil, uridine, guanidine, adenine and adenosine in culture of Paecilomyces hepialid was determined by the developed method of HPLC. The HPLC method was performed on a Waters SunFire C18 (4.6 mm x 250 mm, 5 μm) column with methanol-water gradient elution as the mobile phase. The detection wavelength was 260 nm and the colunmn temperature was controlled at 30 °C. The linear range was 10.00-200.00 mg · L(-1) (r = 0.9994) for uracil, 10.10-202.00 mg · L(-1) (r = 0.9992) for uridine, 10.00-200.00 mg · L(-1) (r = 0.9991) for guanidine, 10.30-206.00 mg · L(-1) (r = 0.9992) for adenine and 10.45-209.00 mg · L(-1) (r = 0.9991) for adenosine, respectively. The RSD of precision was 0.032%, 0.035%, 0.039%, 0.049%, 0.00080%, respectively. The average recoveries of uracil, guanidine, adenine, and adenosine were 97.34%, 99.10%, 101.6%, 98.61% and 100.2% with RSD of 1.3%, 2.1%, 0.96%, 0.95%, and 1.3% respectively. The method showed high sensitivity, good selectivity, linearity and repeatability, which was suitable for the content analysis of 5 nucleosides components in P. hepialid and its extracts.
Chromatography, High Pressure Liquid ; methods ; Nucleosides ; analysis ; Paecilomyces ; chemistry

Objective To detect the expressions of miR-155,T helper type 17 (Thl7) cells,and Th17 cellspecific transcription factor RORγt and effector cytokine interleukin (IL)-17 in peripheral blood and skin lesions from,and to evaluate their relationship in,patients with atopic dermatitis (AD).Methods Peripheral blood was obtained from 37 patients with AD and 33 age-and sex-matched healthy controls,and biopsy specimens from the lesional and perilesional skin of five patients with severe AD as well as from the normal skin of five healthy human controls.Real-time fluorescence-based reverse transcription (RT)-PCR was employed to measure the mRNA expression levels of miR-155,RORγt and IL-17 in peripheral blood mononuclear cells (PBMCs) and skin specimens,flow cytometry to detect the percentage of Th17 cells in PBMCs,enzyme-linked immunosorbent assay (ELISA) to determine the plasma concentration of IL-17.Statistical analysis was done using independent sample's t test,one-way analysis of variance followed by the least significant difference test,and linear correlation analysis.Results Compared with the healthy controls,the patients with AD showed a significant increase in Th17 cell percentage (1.78％ ± 0.52％ vs.0.47％ ± 0.15％,P＜ 0.01),mRNA expression levels of miR-155 (5.78 ± 1.78 vs.1.82 ± 0.46,P＜ 0.01),RORγt (6.08 ± 1.04 vs.1.64 ± 0.52,P＜ 0.01) and IL-17 (7.09 ± 1.75 vs.1.71 ± 0.46,P＜ 0.01),as well as in the plasma concentration of IL-17 ((2.51 ± 6.15) pg/ml vs.(11.80 ± 2.24) pg/ml,P＜ 0.01).There was a sequential decrease in the expression levels of miR-155,RORγt and IL-17 mRNA from lesional skin,perilesional skin to normal skin (F =41.803,17.040 and 37.064 respectively,all P ＜ 0.01).The miR-155 mRNA expression level in PBMCs was positively correlated with the SCORing Atopic Dermatitis (SCORAD) index,Th17 cell percentage,RORγt and IL-17 mRNA expression levels as well as IL-17 plasma concentration (r =0.405,0.426,0.402,0.410 and 0.408 respectively,all P ＜ 0.05).Similarly,the miR-155 expression level was positively correlated with RORγt and IL-17 mRNA expression levels in lesional and paralesional specimens (r =0.428 and 0.435 respectively,both P ＜ 0.05).Conclusion The up-regulated expression of miR-155,Th17 cells and their effector cytokine IL-17 may be associated with the development of AD.

OBJECTIVETo investigate the effects of volatile oils of Rhizoma Acori Tatarinowii (RAT), Semen Myristicae (SM) and Pericarpium Citri Reticulatae (PCR) on percutaneous penetration of bullatine A via hairless mouse skin in vitro.

METHODBy an improved Franz diffusion, the effects of three kinds of volatile oils on the percutaneous penetration of bullatine A were observed and compared with Azone, and the cumulative amount of bullatine A was determined by HPLC.

RESULTThe penetration enhancement ratios of bullatine A with 7% volatile oil RAT and SM, 5% volatile oil of PCR and 3% Azone were 6.52, 6.74, 2.18, 6.03, respectively.

CONCLUSIONThe volatile oil of RAT, SM and PCR enhance permeation of bullatine A, effectively.

Administration, Cutaneous ; Animals ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; pharmacology ; Male ; Mice ; Mice, Nude ; Oils, Volatile ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Skin ; drug effects ; metabolism ; Skin Absorption ; drug effects

OBJECTIVETo investigate the function of apoptosis in esophageal cancer cells induced by soybean isoflavone, and the relation between this apoptosis and expression of bcl-2 and bax.

METHODSIn vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscope and TUNEL staining method were used to quantitatively and qualitatively detect the apoptosis status of esophageal cancer cell line EC-9706 before and after the soybean isoflavone treatment. Immunohistochemical staining and reverse transcription-polymerase chain reaction were used to detect the expression of apoptosis-regulated gene bcl-2 and bax.

RESULTSSoybean isoflavone inhibited the growth of esophageal cancer cell line EC-9706 in a dose- and time-dependent manner. Soybean isoflavone induced EC-9706 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation by transmission electron microscope and staining positive cells, using TUNEL assay. Soybean isoflavone reduced the expression of apoptosis-regulated gene bcl-2, and improving the expression of apoptosis-regulated gene bax.

CONCLUSIONSoybean isoflavone seemed to be able to induce the apoptosis in esophageal cancer. This type of apoptosis might be mediated by down-expression of apoptosis-regulated gene bcl-2 and up-expression of apoptosis-regulated gene bax.

Apoptosis ; drug effects ; genetics ; Cell Line, Tumor ; drug effects ; Dose-Response Relationship, Drug ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Humans ; In Situ Nick-End Labeling ; Isoflavones ; pharmacology ; Proto-Oncogene Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Soybeans ; chemistry ; Time Factors ; bcl-2-Associated X Protein

The purposes of the present study were to investigate the inhibitory effect of quercetin (QUE) preconditioning on endoplasmic reticulum stress (ERS) inducer tunicamycin (TM)-induced apoptosis in RAW264.7 macrophages and the underlying molecular mechanisms. RAW264.7 cells were pretreated with different concentrations (20, 40, and 80 μmol/L) of QUE for 30 min and then treated with TM (5 mg/L) for 12 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC apoptosis detection kit, respectively. The nuclear translocation of activating transcription factor 6 (ATF6) in cells was detected by immunofluorescence analysis and Western blot. Protein and mRNA expressions of C/EBP homologous protein (CHOP) and Bcl-2 were examined by Western blot and real-time PCR, respectively. The results showed that TM reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of TM were significantly inhibited by QUE pretreatment at the concentrations of 40 and 80 μmol/L. Interestingly, we found that QUE also significantly suppressed the TM-induced translocation of ATF6, an ERS sensor, from the cytoplasm to the nucleus. In addition, exposure of RAW264.7 macrophages to TM resulted in a significant increase of the expression of CHOP, a transcription factor regulated by ATF6 under conditions of ERS, as well as a decrease of Bcl-2 at transcript and protein levels. QUE blocked these effects in a dose-dependent manner. These data indicate that QUE can protect RAW264.7 cells from TM-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ATF6-CHOP signaling pathway.
Activating Transcription Factor 6 ; metabolism ; Animals ; Apoptosis ; Cell Survival ; Endoplasmic Reticulum Stress ; Macrophages ; cytology ; drug effects ; Mice ; Quercetin ; pharmacology ; Transcription Factor CHOP ; metabolism ; Tunicamycin ; pharmacology

The infection status of anisakid larvae was examined in 290 marine fish of 25 species and in 108 cephalopods of 3 species purchased in Bayuquan region, Yingko city nearby the coast of the Bohai Sea from may to August 1992. A total of 7,327 larvae were collected from 156 fish of 19 species and 8 squids of one species. The 3rd-stage larvae of Anisakis simplex were collected from 121 fish (63.4%) of 15 species (N = 191) and from 8 squids (14.8%) of one species (N = 54), and they were total, 5,992 (81.8%). Out of remaining 1,335 larvae, 154 (2.1%) were classified as Thynnascaris type B from 23 fish of 4 species, 1,013 (13.8%) as Thynnascaris type C from 79 fish of 13 species. 164 (2.2%) as Hysterothylacium China type V from 20 fish of 4 species, 3 (0.04%) as Raphidascaris from 3 fish of 2 species and one was Pseudoterranova decipiens larva.
Animal ; Anisakiasis/veterinary* ; Anisakiasis/parasitology ; Anisakiasis/epidemiology ; Anisakis/isolation & purification ; Anisakis/classification* ; China ; Fish Diseases/parasitology* ; Fish Diseases/epidemiology ; Fishes ; Larva ; Seawater ; Squid/parasitology*

OBJECTIVETo investigate the mechanism of plasma bilirubin level increase after hepatic ischemia-reperfusion in rats.

METHODSRats were divided into a sham operation group (A group), a 20 min ischemia-reperfusion group (B group) and a 35 min ischemia-reperfusion group (C group). Study time points were 6 hours and 1, 3, and 5 days after the reperfusion. Pathological changes in the livers were studied with histological slides stained with hematoxilin and eosin. Routine biochemistry methods were used to detect the bilirubin level of blood plasma and the bile drained from the ischemic hepatic lobes. RT-PCR was used to analyze the expression of the multidrug resistance-associated protein 2 (MRP2) and mRNA. Immunohistochemistry was used to analyze the localization of MRP2 in the canalicular membrane.

RESULTSB and C groups showed a mild inflammatory reaction without hepatocyte necrosis. At 6 h and 1 day after reperfusion, there was a significant increase of the plasma bilirubin level and a decrease of the bilirubin level of the drained bile in B group. These changes lasted to the day 3 and day 5 in C group. MRP2 mRNA down-regulation was found at 6 h only in the B and C groups. No localization of MRP2 in the canalicular membrane was found but it appeared in "esicules" under the canalicular membrane in C group.

CONCLUSIONSAbsence of MRP2 localization in the canalicular membrane could be the cause of the blood plasma bilirubin level increase after liver ischemia-reperfusion.

Animals ; Bilirubin ; blood ; Liver Diseases ; blood ; Male ; Multidrug Resistance-Associated Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; blood

OBJECTIVETo explore the effect of retinoblastoma binding protein 2 (RBP-2), a histone H3K4 demethylase, on osteogenic differentiation of human adipose-derived stromal cell (hASC).

METHODSAccording to the GenBank sequence information of RBP-2, four different small interfering RNAs (siRNA) targeting RBP-2 gene were designed and the corresponding short hairpin RNAs (shRNA) were cloned into pLL 3.7 lentivirus RNA interference vector. The lentivirus with RBP-2-siRNA was packaged in 293T cells. The effective sequence was examined and selected by Western blotting and real-time PCR. The lentiviruses with efficient knockdown effects were used to infect hASC. On the 14th day after osteogenic differentiation, alkaline phosphatase (ALP) activities of hASC were quantitatively tested and at the same time, ALP staining and alizarin red staining were performed to assess the difference of osteogenic differentiation between the knockdown group and the control group.

RESULTSThe recombinant lentivirus siRNA targeting RBP-2 was successfully constructed and the expression of RBP-2 mRNA and protein were dramatically suppressed by infection with RBP-2-siRNA lentivirus. On the 14th day after osteogenic induction, ALP activity of hASC in the knockdown group [(299.2 ± 22.7), (224.3 ± 17.7) U/g] was much stronger than that in the control group [(129.9 ± 12.9) U/g, P < 0.05] and the same result was achieved for the ALP staining and alizarin red staining.

CONCLUSIONSThe constructed RBP-2-siRNA lentivirus could markedly decrease the expression of RBP-2 and promote osteogenic differentiation of hASC. It indicated that RBP-2 can repress the osteogenic differentiation of hASC.

Adipose Tissue ; cytology ; Adult ; Alkaline Phosphatase ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Cells, Cultured ; Female ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Lentivirus ; Osteogenesis ; Osteosarcoma ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Retinoblastoma-Binding Protein 2 ; genetics ; metabolism ; Stromal Cells ; cytology ; metabolism

OBJECTIVETo screen the proteins interacting with FXR1P for functional investigation of FXR1P.

METHODSThe yeast strain AH109 transformed with the recombinant expression vector pGBKT7/FXR1 was mated with the yeast strain Y187 pretransformed with human fetal brain cDNA library. The positive clones were screened and identified by sequence analysis.

RESULTSThe recombinant expression vector pGBKT7/FXR1 was constructed successfully. Five proteins binding to FXR1P were screened from human fetal brain cDNA library using the yeast two-hybrid system, including CMAS, FTH1, GOLGA4, HSD17B1 and CSH1.

CONCLUSIONSThese results provide new clues for investigating the biological functions of FXR1P and the pathogenesis of Fragile X syndrome.

Autoantigens ; genetics ; metabolism ; Estradiol Dehydrogenases ; genetics ; metabolism ; Ferritins ; genetics ; metabolism ; Gene Library ; Humans ; Membrane Proteins ; genetics ; metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; genetics ; RNA-Binding Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

Eight alkaloids were isolated from the thin sulfuric acid extracts of the fresh roots of Stephania dentifolia by aluminum oxide, silica and Sephadex LH-20 column chromatography methods. Based on the spectroscopic analysis and chemical evidence, the structures of these alkaloids were identified as sinoacutine (1), sinomenine (2), cephamonine (3), tetrahydropalmatine (4), capaurine (5), stepharanine (6), (+)-stepharine (7) and palmatine (8). All compounds were obtained from this plant for the first time.
Alkaloids ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Plant Roots ; chemistry ; Stephania ; chemistry