Objective To establish a HPLC/MS/MS method for the determinati on of gentiopicroside in human urine.Methods The urine sample was treated by solid-phase extraction with internal standard of caffeine;The RESCEK C8 colum n(150mm?2.1mm,5 ?m) was used as the analytical column with a mobilep hase consisting of methanol-10mmol? L-1 NH4AC buffer(pH=6.5)-acetonit rile(50∶40∶10,V/V),the flow rate was 0.2 mL? min-1;A triple quadruple tandem mass spectrometer was used as the detector,Electrospray ioniza tion source was applied and operated in positiveion mode.Gentiopicroside and caffeine were detected by monitoring the ion transition of m/z 374.1→ 195.2 and m/z 195.2→ 138.2 respectively.Results The linear range was 30～ 9000 ng?mL-1(r=0.9980) for gentiopicroside in human urine.The recovery was 91.10% ～ 9 6.21 %,The absolute recovery was 100.52% ～ 103.83%,The within-day and between-day precisions were less than 10 %.Conclusion The method is proved to be sensitive,accurate,rapid,specific.

To study the sesquiterpenoid constituents in the whole plant of Sarcandra glabra, silical column chromatography, Sephadex LH-20, reverse phase ODS column chromatography and preparative HPLC were used to isolate 70% EtOH extract of Sarcandra glabra. The structures were elucidated based on spectroscopic data (HR-ESI-MS, 1H NMR, 13C NMR, HSQC, HMBC and NOESY). Four sesquiterpenoids were obtained and identified as 4alpha-hydroxy-5alphaH-lindan-8 (9)-en-8, 12-olide (1), chloranthalactone E (2), 8beta, 9alpha-dihydroxylindan-(5), 7 (1)-ieb-8alpha, 12-olide (3) and chloranoside A (4), respectively. Compound 1 is a new sesquiterpene lacone.

Objective To study the biodistribution of anti-HER-2/neu monoclonal antibody Herceptin labeled by 131I(131I-Herceptin) in healthy KM mice and nude mice bearing human ovarian cancer xenografts and radioimmunoimaging (RII) of the nude xenografts-bearing mice.Methods 131I-Herceptin was prepared using Iodogen method.The labeling efficiency, radiochemical purity, stability and immunocompetence were measured.The percentage activity of injection dose per gram of tissue (%ID/g) and the radioactivity ratio of tumor to non-tumor tissue (T/NT) were calculated for each time point.The optimal time for imaging was investigated by comparing the 131I-Herceptin SPECT for the nude mouse models bearing ovarian cancer xenografts at different time points.Results The labeling efficiency and radiochemical purity of 131I-Herceptin were 89.8% and 98.4%, respectively.The labeling was stable and had good immunocompetence.131 I-Herceptin was cleared rapidly mainly through liver, spleen and kidneys, consistent with first order two-compartment model.The uptake of 131I-Herceptin in the tumors bearing human SKOV-3 xenografts was much higher than that in nontumor tissue.The% ID/g was 18.08 in the tumor at 24 h post injection.The T/NT ratio increased with time and was 27.27 at 72 h post injection.The tumors in nude mice bearing SKOV-3 xenografts could be visualized on 131I-Herceptin SPECT imaging 2 h post injection; definitely identiffed 48 h post injection and the radioactivity ratio of tumor to contralateral tissue was 11.44 at 120 h post injection.However, the tumor in nude mice bearing HO-8910 xenografts did not show abnormal uptake of 131 I-Herceptin at each time point.Conclusions 131 I-Herceptin is a good radiopharmaceutical targeting SK-OV-3 xeuografts and it may be useful in imaging carcinoma of ovary and target therapy of its metastases with high HER-2/neu expression.

Objective Acquiring genetic information of Y-SNPs and Y-STRs genetic makers from samples with the surname of Kong, which is useful for exploring the correlation between surname and Y chromosome in forensic applications studies.Methods Two multiplex genotyping assays and SNaPshot assay were used to analyze 255 unrelated male blood samples who share the same surname Kong and 330 unrelated male blood samples obtained randomly. 17 Y-STRs were typed for the surname Kong population samples. The software Arlequin 3.5.1.2 and the program Network 4.6.1.1 were used for data statistical analysis.Results 13 haplogroups were observed. The highest haplogroup frequency in the two populations were O3a2c1a-M117 (21.57%, 14.85%). 196 haplotypes in Kong population deifned by 17 Y-STRs locus were obtained and the haplotype diversity was 0.9939. 14-12-25-28-19-15-12-19-12-11-12-22-12-11-14-10-19 is the typical haplotype. Median Joining algorithm and Mismatch Distribution were adopted to analyze the Y-STR haplotye under haplogroup O3-M122, and the result shows that there are two “central star” distribution. Conclusion Combined with Y-SNP and Y-STR analysis showed that the Kong population had experienced complicated exchanges and expansion or continued growth, which has more than one surname origin. Hence, its population genetic structure and historical differences have potential applications in forensic science.

In order to study the excretion of genistein (GEN) capsule, an estrogen drugs, in human, 30 healthy volunteers were selected and orally administered 50, 100, and 300 mg genistein in an parallel study. Genistein were determined in urine by LC-MS/MS and glucuronidated genistein (GENG) were indirectly determined with enzymatic hydrolysis in urine by LC-MS/MS, and the pharmacokinetic parameters were analyzed by DAS software (ver 2.0). The result showed that the concentrations of genistein in human urine were less than 1% of the GENG, and the cumulative excretion of GEN in 48 h were 0.037, 0.134, and 0.142 mg, separately, and the urinary excretion percentage were only 0.07%, 0.13%, and 0.05%, separately. But the cumulative excretion of GENG in 48 h was 5.3, 13.8, and 15.4 mg, separately, and the urinary excretion percentage were 10.6%, 13.8%, and 5.1%, separately, and the max urinary excretive rate was 0.4, 1.0, and 1.4 mg x h(-1), separately (tmax were 6 h). Studies showed that part of drug excreted through kidney in a form of GENG in human, and the cumulative urinary excretion and the maximum excretion rate of GENG showed a proportional increase conditioned with the dose in the range of 50-100 mg, but showed non-linear increase feature in 300 mg.

Animals ; Male ; Particle Size ; Rats ; Rats, Sprague-Dawley ; Respiratory Distress Syndrome, Adult ; chemically induced ; Silicon Dioxide ; chemistry ; toxicity

Background There is no effective method to regenerate the optic nerve after injury. It has been recently reported that α-crystallin could promote the survive rate and axon regeneration of retinal ganglion cells (RGCs) effectively. However,the molecular mechanism is not clear. Objective This study was to identify the site of RGCs where the exogenous α-crystallin bind to. Methods RGCs was isolated from retinas of two 2-day-old Long Evans rats and primarily cultured. The positive rate of the RGCs was assessed by counting the number of positive cells for fluorescently-labeled thy1. 1 and cy3 under the fluorescence microscope. The biotinylated exogenous α-crystallin was evaluated by direct coloration and the activity of molecular chaperones was measured by insulin test.After identifying the success of biotinylation along with the activity of molecular chaperones,biotinylated α-crystallin was co-incubated with RGCs and the cells then were reacted to fluorescently labeled avidin for the observation of binding site of exogenous α-crystallin under the laser confocal microscope. Results RGCs of 94% were survived through primary culture. The coloration of biotinylated α-crystallin labeled by the direct coloration method was more intensive, and the value of A450 descended as the decrease of biotinylated α-crystallin concentration,indicating that the α-crystallin was biotinylated successfully. The activity of molecular chaperones of biotinylated α-crystallin was significantly strong but no significant change after being biotinylated after co-incubation of RGCs with biotinylated α-crystallin. Laser confocal microscope examination revealed that co-incubated RGCs with biotinylated α-crystallin showed the red fluorescence on membrane and axon of RGCs rather than cytoplasm and nucleus. The absent response was seen in the control group. Conclusion Exogenous α-crystallin can specifically combine with the membrane of RGCs to play the biological function,but its binding mode and mechanism need further study.

Mycoplamas are a group of wall-less prokaryotes widely distributed in nature, some of which are pathogenic for humans and animals. There are many lipoproteins anchored on the outer face of the plasma membrane, called lipid-associated membrane proteins (LAMPs). LAMPs are highly antigenic and could undergo phase and size variation, and are recognized by the innate immune system through Toll-like receptors (TLR) 2 and 6. LAMPs can modulate the immune system, and could induce immune cells apoptosis or death. In addition, they may associate with malignant transformation of host cells and are also considered to be cofactors in the progression of AIDS.
Animals ; Humans ; Lipids ; Membrane Proteins ; metabolism ; Mycoplasma ; physiology ; Mycoplasma Infections ; immunology ; metabolism ; microbiology ; pathology ; Protein Binding ; Signal Transduction

Objective To investigate the anti-apoptotic function of clusterin in LNCaP cell line and the role of clusterin antisense oligodcoxynucleotide(AS-ODN)in TNF-?-induced death of LNCaP cell. Methods The wild type LNCaP(group L),LNCaP transfected with the control vector(group M),LNCaP transfected with full-length clusterin expression vector(group A,ie,study group)were cultured.For the de- tection of cytotoxic effect of TNF-?,MTT and ELISA methods were used to determine the cell proliferation and apoptosis of the 3 clones,and the changes of proliferation and apoptosis in A cell after transfection of clusterin AS-ODN were also assessed.Results MTT method showed that the cell proliferation activity(A value)of groups L,M,and A were 0.84?0.03,0.85?0.04,0.95?0.03,respectively;the difference be- tween groups L and M was not significant(P＞0.05);but compared with group A the cell proliferation activ- ity was significantly lower in groups L and M(P＜0.01 for both).ELISA resuhs showed that the A values of groups L,M,and A were 0.59?0.04,0.62?0.03,0.33?0.04,respectively;the difference between groups L and M was not significant(P＞0.05);but compared with group A,the apoptosis rates were significantly higher in groups L and M(P＜0.01 for both).In group A,A values of cell proliferation activity in subgroups control,AS-ODN,TNF-?,TNF-?+AS-ODN were 1.30?0.03,1.25?0.03,0.99?0.03,0.80?0.03, respectively;the differences between each group were significant(P＜0.05 for all).And the A values of cell apoptosis in the above 4 groups were 0.02?0.00,0.21?0.02,0.63?0.07,1.16?0.04,respectively,the differences between each group were significant(P＜0.01 for all).Conclusions Stable transfection and subsequent expression of clusterin result in resistance to the cytotoxic effect of TNF-?.Transfection with clus- terin AS-ODN enhances cytotoxic effect of TNF-?in A cells.These results suggest that clusterin plays an im- portant role in anti-apoptotic function in LNCaP cell line.

OBJECTIVETo study the effects of licorice on the proliferation of intestinal crypt stem cell line IEC-6 and the expression of p53.

METHODSInduced by difluoro-methylornithine (DFMO), polyamine-depleted IEC-6 cells under growth inhibition were used as the pathological cell model in this study. Cells were divided into four groups, i. e., the control group, the DFMO-treated group, the high dose licorice group, and the low dose licorice group. The control group consisted of IEC-6 cells cultured in normal condition. The other three groups were all treated with 5 mmol/L DFMO. The high dose and low dose licorice groups were supplemented with 40 and 80 microg/mL licorice granule respectively. All the groups were cultured for 6 successive days. The cell number and viability were determined using flow cytometry. The level of p53 protein was detected by Western blot. The p53 mRNA levels and stability were detected using fluorescent quantitative Real-time PCR.

RESULTSCompared with the control group, the cell growth of the DFMO group was obviously inhibited on the 4th day (P < 0.05). The cell number increased more obviously in the low dose licorice and the high dose licorice groups in a dose-dependent way on the 6th day when compared with the DFMO group (P < 0.05). When compared with the control group, significantly elevated expression levels of p53 protein and mRNA in cells of the DFMO group were detected after 6-day treatment (P < 0.05). When compared with the DFMO group, the expression levels of p53 protein and mRNA were significantly down-regulated in the low dose licorice and the high dose licorice groups (P < 0.05). The degradation of p53 mRNA was the fastest in the control group, while the degradation speed of cells in the DFMO group was the slowest.

CONCLUSIONOne of mechanisms for protective and healing effects of licorice on the intestinal mucosa was possibly through down-regulating the stability of p53 mRNA, lowering the expression of p53, thus promoting the proliferation of the intestinal crypt stem cells.

Animals ; Cell Line ; Cell Proliferation ; Glycyrrhiza ; Intestines ; cytology ; metabolism ; RNA Stability ; drug effects ; RNA, Messenger ; genetics ; Rats ; Stem Cells ; cytology ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; metabolism