OBJECTIVETo investigate the effect of tBHQ and sulforaphane on the protein expression in Nrf2-ARE signaling pathway of Caco2 cells.

METHODSHuman colorectal carcinoma Caco2 cells were treated with 20 micromol/L tBHQ and 5 micromol/L sulforaphane (SFN) respectively. Real time PCR, Western blotting and immunoflourescence staining (IF) were performed to measure the target gene expression.

RESULTSNrf2, AKR1C1 and NQO1 protein expressions were increased time-dependently in Caco2 cells after treatment with tBHQ and SFN. Time-course experiments showed that tBHQ and SFN increased the accumulation of Nrf2, and concomitantly increased the protein levels of AKR1C1 and NQO1. Real-time PCR and Western blotting showed that tBHQ and SFN significantly increased the expression of Nrf2 at 8h after the treatment, and AKR1C1 and NQO1 at 16 h. Confocal microscopy technique showed that Nrf2 accumulated in the nucleus at 6-8 h after treatment with tBHQ. After 1 h treatment with tBHQ the nuclear Nrf2 maintained at elevated level for at least 4 h with tBHQ withdrawn.

CONCLUSIONtBHQ and SFN induced nuclear accumulation of Nrf2 and activated Nrf2-dependent regulation of ARE-mediated gene expression in Caco2 cells. In addition, the results provide experimental evidence for choosing the dose and frequency of the inducer in cancer chemoprevention study and in developing inhibitors of Nrf2-ARE signaling pathway.

Anticarcinogenic Agents ; pharmacology ; Antioxidants ; metabolism ; pharmacology ; Caco-2 Cells ; Calcium-Transporting ATPases ; antagonists & inhibitors ; Humans ; Hydroquinones ; pharmacology ; Isothiocyanates ; NF-E2-Related Factor 2 ; genetics ; metabolism ; physiology ; Oxidative Stress ; genetics ; physiology ; Response Elements ; physiology ; Signal Transduction ; drug effects ; Thiocyanates ; pharmacology

A full-length cDNA encoding a MYB-related regulatory gene was isolated from a cDNA library prepared from mRNAs of the red line callus of S. medusa by TD-PCR. The cDNA, designated SmP, is 969 nucleotides long and has an open reading frame of 771 bp with a deduced amino acid sequence of 256 residues. The putative protein of SmP has two typical conversed R2R3-Myb DNA-binding domains in N-terminal and displays a rather high degree of similarity to OsMYB from rice and LBMI from tobacco, showing 73% and 70% identity within the DNA-binding domains. However, the C-terminal domain of the SmP protein does not show obvious similarity to any other known protein sequence. It is rich in hydrophilic amino acids, especially in serine residues (18.38%), partly organized in homopolymeric stretches, a feature often found in activation domain of transcription factors.
Amino Acid Sequence ; Cloning, Molecular ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; classification ; genetics ; metabolism ; Polymerase Chain Reaction ; Saussurea ; classification ; genetics ; metabolism ; Sequence Homology, Amino Acid ; Transcription Factors ; chemistry ; genetics ; metabolism

Objective To investigate the clinical effect of S-1 combined with oxaliplatin (SOX regimen) in treatment of the patients with advanced pancreatic cancer. Methods A total of 106 patients with advanced pancreatic cancer in Qingdao Fuwai Hospital from April 2015 to June 2017 were randomly divided into the treatment group (53 cases) and the control group (53 cases) according to the random number table method. Patients in the control group were treated with S-1 combined with cisplatin treatment, and patients in the treatment group were treated with SOX regimen. The cell proportion of CD3+, CD4+, CD8+and CD4+/CD8+before treatment and after 2 cycles of treatment were detected by using flow cytometry of both groups. The clinical curative effects, immunity and adverse reactions of both groups were compared by usingχ2 test and t test. Kaplan-Meier method was used to make the survival analysis. Results After two cycles of treatment, there were 4 cases of complete remission (CR), 23 cases of partial remission (PR), 17 cases of stable disease (SD), 9 cases of progression disease (PD) in the treatment group, and 0 case of CR, 18 cases of PR, 20 cases of SD, 15 cases of PD in the control group. The rate of CR+PR in the treatment group was higher than that in the control group [50.94％(27/53) vs. 33.96％(18/53)], and there was a statistical difference (χ2=5.936, P<0.05). There was a significant difference in the cell proportion of CD4+and ratio of CD4+/CD8+between the two groups before and after treatment [the treatment group: (27.31±2.48)％ vs. (37.05±2.53)％, χ2= 6.491,P< 0.01; 0.91 ±0.23 vs. 1.53 ±0.50, χ2 = 5.913, P< 0.01; the control group: (27.43 ±2.47)％ vs. (30.32 ± 2.41)％,χ2= 11.214, P<0.01; 0.90±0.22 vs. 1.22±0.34,χ2=7.992, P<0.01]. After 2 cycles of treatment, the cell proportion of CD4+and ratio of CD4+/CD8+of the treatment group were higher than those of the control group, and there were statistical differences (χ2=5.309, P<0.01;χ2= 7.112, P< 0.01). The incidence rate of side effects had no significant difference in both groups after two cycles of treatment [22.64％ (12/53) vs. 18.87％ (10/53), χ2= 1.924, P> 0.05]. The progression-free survival time in the treatment group was longer than that in the control group (P<0.05). Conclusions SOX regimen has a favorable effect on the patients with advanced pancreatic cancer. It can help to improve the immunity and prolong the survival time of the patients.

Anaphylaxis is increasingly in children, which is currently undernotified, underdiagnosed, and undertreated in China.In order to further improved the understanding and management of anaphylaxis, this issue reviews the pathogenesis, triggers and risk factors, clinical diagnosis and management of anaphylaxis, thus offers the recommedations of anaphylaxis in Chinese children based on previous published evidence-based guidelines and practice parameters.Recommendation aims to develop guiding principles for the diagnosis and management of anaphylaxis in children, and provide a framework for the development of new guidelines.

Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.
Animals ; Base Sequence ; Green Fluorescent Proteins ; genetics ; HeLa Cells ; Humans ; Introns ; genetics ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Mutation ; RNA, Catalytic ; genetics ; RNA, Messenger ; genetics ; Tetrahymena ; enzymology ; Trans-Splicing ; Transcription, Genetic

OBJECTIVETo explore in-vivo targeted imaging techniques for liver cancer detection using quantum dots (QDs) labeled probes in a nude mouse model of human hepatocellular carcinoma.

METHODSMercaptoacetic acid (MAA) modified QDs were linked to mouse-anti-human alpha-fetoprotein (AFP) monoclonal antibody to form water soluble QD-AFP-Ab probes, which were validated by spectra analyses and transmission electron microscope. The probes were firstly used to detect AFP antigen in human hepatocellular carcinoma cell line HCCLM6 in-vitro by one-step immunofluorescence method. In-vivo tumor xenografts and lung metastases models were then established by inoculation of HCCLM6 cells subcutaneously and into the tail vein of nude mice, respectively. QD-AFP-Ab probes were injected into the tail vein of the tumor bearing mice for live animal fluorescence imaging. Spectra of tumor and normal tissue were analyzed under illumination of Ti: sapphire laser. Serum levels of alanine amino transferase, aspartate amino transferase, blood urea nitrogen and creatinine were determined by conventional biochemical analysis. The liver, spleen, lungs, kidneys, heart and brain of the experimental nude mice were investigated for nonspecific uptake of the probes by confocal microscope.

RESULTSThe QD-AFP-Ab probes had broad excitation spectra and high fluorescence intensity. They could specifically and efficiently recognize AFP antigen in hepatocellular carcinoma cells. Tumor targeting imaging using these probes were successful without any acute toxicity to the experimental animals. Spectra analysis showed that the probes per field were lower in the centre than the periphery of the tumor. Non-specific uptake of QD-AFP-Ab probes occurred mainly in the liver, spleen and lungs.

CONCLUSIONSQD-AFP-Ab probes have good optical properties and biocompatibility for in-vivo targeted imaging of hepatocellular carcinoma. Such approach promises to be highly desirable for molecular targeted research of liver cancer.

Animals ; Antibodies, Monoclonal ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Diagnostic Imaging ; methods ; Fluorescent Antibody Technique ; methods ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Lung Neoplasms ; metabolism ; secondary ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microscopy, Fluorescence ; Molecular Probes ; metabolism ; pharmacokinetics ; toxicity ; Neoplasm Transplantation ; Quantum Dots ; Tissue Distribution ; alpha-Fetoproteins ; immunology ; metabolism

During 2009-2012, the Nam Dinh virus (NDiv) was detected from the samples of Culex pipiens quinquefasciatus in Shenzhen China. In this study, cell culture,SYBR Green I based real time RT-PCR and RT-PCR were performed to analyze the cell susceptibility and other biological characteristics of the NDiV isolates. The results showed that C6/36 cell line was susceptible to four isolates of Culex pipiens quinquefasciatus. The "S" type amplification curve and specific melting curve were obtained in the realtime fluorescence quantitative RT-PCR based on SYBR Green I for the detection of the NDiV from the mosquito. The target bands from the RdRp gene and partial fragment of ZmHel1 gene were observed using agarose gel electrophoresis. Both the nucleotide and amino acid sequences of four Shenzhen isolates showed more than 99.00% homology with the Vietnam representative NDiV strain (02VN178). Phylogenetic analysis showed that four Shenzhen isolates shared the same evolution branch as the Vietnam representative NDiV strain. This is the first report of NDiV in China.
Animals ; China ; Culex ; virology ; Nidovirales ; classification ; genetics ; isolation & purification ; Phylogeny

OBJECTIVETo explore the impact of abnormal myoelectricity at gastroduodenal anastomosis on gastric emptying in rats.

METHODSRats were randomly divided into experimental group (n=16) and control group (n=16). Pylorectomy and end-to-end gastroduodenal anastomosis were performed in the experimental group and electrodes were implanted in the serosal surface adjacent to the anastomosis. Slow waves were recorded by the implanted electrode in vivo. Gastric emptying was examined by scintigraphy.

RESULTSAt the first week after surgery, antral slow-wave frequency was significantly lower in the experimental group (0.8±1.4 vs. 3.3±1.2, P<0.01), as was the duodenal slow-wave frequency (2.1±0.6 vs. 11.1±0.7, P<0.01). There was no consecutive slow-waves transduction across the pylorus or the anastomosis. Within 12-16 weeks after operation, antral slow-wave frequency in the experimental group and the control group were (8.7±0.6) cpm and (4.0±0.4) cpm, respectively (P<0.01), and duodenal slow-wave frequency were (11.1±0.8) cpm and (10.8±0.7) cpm, respectively (P>0.05). Retrograde and antegrade myoelectricity transduction through the anastomosis were detected. The mean semi-emptying time in the proximal stomach was 14.7 min in the experimental group and 13.6 min in the control group (P>0.05). Radionuclide retention rate was 25.4% in the experimental group and 39.4% in the control group (P>0.05). The mean semi-emptying time in the distal stomach was 25.3 min in the experimental group and 10.5 min in the control group (P<0.01). Radionuclide retention rate was 46.4% in the experimental group and 18.7% in the control group (P<0.01).

CONCLUSIONThe abnormal myoelectricity in the region of gastroduodenal stoma may delay liquid gastric emptying in pylorectomy rats.

Animals ; Duodenum ; physiology ; surgery ; Gastric Emptying ; physiology ; Gastroenterostomy ; Male ; Myoelectric Complex, Migrating ; physiology ; Rats ; Rats, Sprague-Dawley ; Surgical Stomas ; physiology

OBJECTIVETo study the chemical constituents of the roots of Capparis tenera.

METHODThe chemical constituents were isolated and repeatedly purified by kinds of column chromatography and the structures were elucidated by the NMR spectra and physicochemical properties.

RESULTEight compounds were isolated and identified as erigeside C (1), glucosuringic acid (2), vanillic acid 4-O-beta-D-glucoside (3-methoxy 4-glucosyl-benzoic acid) (3), 4-O-beta-D-glucopyranosylbenzoate (4), 3', 5'-dimethoxy- 4-O-beta-D-glucopyranosyl-cinnamic acid (5), tachioside (6), 2, 3-dihydroxy-1-(4-hydroxyl-3, 5-dimethoxyphenyl)-1-propanone (7) and acacetin 7-rutinoside (8).

CONCLUSIONCompounds 1-8 were all isolated from this plant for the first time and the compound 8 was isolated from this gene for the first time.

Capparis ; chemistry ; Chromatography ; Drugs, Chinese Herbal ; chemistry ; Glycosides ; chemistry ; Magnetic Resonance Spectroscopy ; Plant Roots ; chemistry ; Solubility ; Water ; chemistry

OBJECTIVETo investigate the relationship between the dendritic cell (DC) subsets and transcriptive factors, T-bet, GATA-3, and immune imbalance in acquired severe aplastic anemia (SAA).

METHODSThe DC1 (HLA-DR+Lin-CD11c+) and DC2 (HLA-DR+Lin-CD123+) in peripheral blood mononuclear cells (PBMNC) were measured with flow cytometry (FCM), the expressions of T-bet mRNA and GATA-3 mRNA in PBMNC with semiquantitative RT-PCR and the plasma level of IFN gamma and IL-4 with ELISA in 29 SAA patients and 16 healthy controls.

RESULTSThe percentages of DC1 in PBMNC were (0.44 +/- 0.24)% and (0.73 +/- 0.30)% in untreated and recovered SAA patients respectively, both were higher than that in controls (0.29 +/- 0.10)% (P < 0.05). The percentage of DC2 in the untreated cases was lower than that of recovered ones or controls [(0.18 +/- 0.14)% vs (0.28 +/- 0.20)% and (0.29 +/- 0.13)%] (P < 0.05). DC1/DC2 ratios were 3.45 +/- 2.71 and 2.90 +/- 0.95 in untreated and recovered groups respectively, both were higher than that in controls (1.15 +/- 0.56) (P < 0.05). No statistic difference in DC1/DC2 ratio was found between untreated and recovered patients (P < 0.05). The relative mRNA expression levels of transcriptive factor T-bet were 0.37 +/- 0.07, 0.20 +/- 0.07 and 0.17 +/- 0.05 in the above 3 groups, respectively, untreated group being higher than that of recovered group or healthy controls (P < 0.05). There was no statistic difference of GATA-3 expression among the 3 groups (P > 0.05). T-bet/GATA-3 ratio was 0.72 +/- 0.13 in untreated group, being higher than that of recovered group (0.33 +/- 0.08) or controls (0.35 +/- 0.11). The plasma level of IFN gamma in the untreated group was (50.9 +/- 1.1) ng/L, which was higher than that of recovered group [(49.7 +/- 0.9) ng/L] or controls [(49.7 +/- 0.7) ng/L]. There was significant positive correlations between T-bet and DC1/DC2 ratio (r = 0.445, P < 0.01), as well as between T-bet and IFN gamma (r = 0.402, P < 0.01).

CONCLUSIONEither DC1/DC2 or T-bet/GATA-3 ratio might become an index to estimate immune imbalance. High-expressed T-bet was related to the progress of SAA. In patients with SAA, DC1/DC2 ratio returns to normal range later than that of routine blood test does, indicating that immunosuppressive therapy should not be withdrawn too earlier.

Adolescent ; Adult ; Anemia, Aplastic ; blood ; immunology ; Child ; Dendritic Cells ; immunology ; Female ; GATA3 Transcription Factor ; blood ; genetics ; Humans ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Male ; Middle Aged ; RNA, Messenger ; genetics ; T-Box Domain Proteins ; blood ; genetics ; Young Adult