Objective To investigate the effect of simulated microgravity on the proliferation and differentiation of the human megakaryocyte cells in vitro. Methods The fourth generation rotating cell culture system (RCCS-4) was used to generate the simulated microgravity environment. The cell viability was assessed by trypan blue staining method. The proliferation of cells was assessed by cell counting method and CCK8 method. The CD41+/CD61+ cells rate and the cells cycle were detected by flow cytometry. The expression levels of thrombopoietin receptor (c-mpl) and transcription factors were detected with RT-PCR. Results After 24, 48, 72 h, culture under simulated microgravity resulted in a significant decrease in the cell number , proliferative activity, cells in the G2/M phase and levels of c-mpl mRNA expression in comparison with that under the normal gravity (P < 0.05). After 48 h and 72 h culture, CD41+/CD61+ cells ratio decreased and RUNX-1 mRNA expression was down-regulated in cells of the group SMG compared with that of the group NG (P < 0.05). Conclusion Microgravity can inhibit the proliferation and differentiation of human megakaryocyte cells in vitro. The mechanism may be that TPO/c-mpl pathway was inhibited by down regulating the expression of c-mpl which transcriptional inhibition lead to.

This study is to establish an HPLC-DAD-ELSD method for simultaneous determination of 5 flavones and saponins in Rhizoma Anemarrhenae including neo-mangiferin, mangiferin, timosaponin B II, timosaponin B III and timosaponin A III. Samples were analyzed on a Merck Purospher STAR column(4.6 mm x 250 mm, 5 μm). The mobile phase consisted of acetonitrile( A) and 0. 1% formic acid (B) with gradient elution at a flow rate of 1.0 mL · min(-1). The column temperature was set at 40 °C. The DAD detector wavelength was set at 254 nm. The ELSD conditions were as follows: the nebulizing gas flow rate was 2.0 L · min(-1) and temperature of drift tube was 105 °C. The volume was 10 μL. The five compounds were well separated with good linear correlations. The mean recoveries were between 102.0%-104.0%. This method was quick and reliable which provides a foundation for quality control of R. Anemarrhenae.
Anemarrhena ; chemistry ; Chromatography, High Pressure Liquid ; instrumentation ; methods ; Drugs, Chinese Herbal ; analysis ; Flavones ; analysis ; Rhizome ; chemistry ; Saponins ; analysis

OBJECTIVETo construct prokaryotic expression systems of ltB/ctB-ompL1/1 fusion genes and to determine the L.interrogans carrying status in leptospirosis patients with the expression products.

METHODSThe fusion genes ltB-ompL1/1 and ctB-ompL1/1 were constructed using linking primer PCR method. SDS-PAGE was used to examine expression of the target recombinant proteins rLTB-rOmpL1/1 and rCTB-rOmpL1/1. Western blot and GM1-ELISA were used to measure the immunogenic and GM(1)-binding activities of rLTB-rOmpL1/1 and rCTB-rOmpL1/1, respectively. PCR and MAT were performed to detect the expression of ompL1 gene in 97 wild L.interrogans strains. Antibodies against ompL1 gene products in serum samples of 228 leptospirosis patients were detected with ELISA method.

RESULTSThe homogeneity of nucleotide and putative amino acid sequence of ltB-jompL1/1 and ctB-ompL1/1 fusion genes were 99.7 % - 99.9 % and 99.5 % - 100 %, in comparison with the reported corresponding sequences. Expression outputs of both rLTB-rOmpL1/1 and rCTB-rOmpL1/1, mainly present in inclusion body, accounted for 10% of the total bacterial protein. Both rLTB-rOmpL1/1 and rCTB-rOmpL1/1 could combine to rabbit anti-rOmpL1/1 serum and bovine GM(1). 89.7 % of L.interrogans wild strains had ompL1 gene. 87.6% of the wild L.interrogans strains presented positive results for MAT (titers :1:4-1:256) with the rabbit anti-rOmpL1/1 or anti-rOmpL1/2 sera. 86.8% and 88.6% of the patients' serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies, respectively.

CONCLUSIONThe fusion proteins, rLTB-rOmpL1/1 and rCTB-rOmpL1/1, showed high immunogenic and GM(1)-binding activities. ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and its products expressed by different genotypes exhibit extensive cross-antigenicity.

Bacterial Outer Membrane Proteins ; genetics ; immunology ; Bacterial Toxins ; genetics ; Bacterial Vaccines ; genetics ; Cloning, Molecular ; Enterotoxins ; genetics ; Escherichia coli Proteins ; genetics ; Genes, Bacterial ; genetics ; Humans ; Leptospira interrogans ; genetics ; immunology ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccines, Synthetic ; genetics

OBJECTIVETo determine the effects of leptospiral strains with different virulence on intracellular free Ca(2+)level and its relation with phospholipase C (PLC) activity of L.interrogans.

METHODSL.interrogans-j infection cell modals were established with Vero and J774A.1 cell lines. Vero and J774A.1 cells were co-incubated with L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 (strong virulence) and serogroup Pomona serovar pomona strain 56608 (weak virulence) and L.biflexa serogroup Samaranga serovar patoc strain Patoc I (non-virulence). Intracellular free Ca(2+)levels were detected by laser scanning confocal microscopy with specific fluorescence labeling of fluoj3/AM. Using [(3)H] PIP2 as the substrate, the PLC activities in the culture supernatant, and cytoplasma and cytomembrane of the three strains of Leptospira were measured by isotope assay.

RESULTSThe baseline intracellular free Ca(2+)levels in the normal Vero and J774A.1 cells were (102.3+/-8.2)% and (105.9+/-7.3)%,respectively. The fluorescence intensity in the two cell lines incubated with L.biflexa strain Patoc I were fluctuated in range of (102.3+/-8.2)%approximate, equals(102.2+/-8.3)% during the observation period. The intracellular free Ca(2+)levels in the two cell lines infected with L.interrogans strain 56601 showed elevation with double peak patterns, with first peaks of (430.5+/-35.7)%, (747.5+/-18.5)% and the second peak of (380.6+/-17.4)%, (804.6+/-22.4)%, respectively. When the cells were infected with L.interrogans strain 56608, the intracellular free Ca(2+)levels were rising slowly with a single slope-like pattern, with the maximal of (235.0+/-19.3)% in Vero cells and (402.4+/-17.4)% in J774A.1 cells, which were significantly lower than those in the cells infected with L.interrogans strain 56601 (P<0.01). The culture supernatants, and cytoplasma and cytomembrane proteins of all three strains displayed PLC activities (P<0.05).

CONCLUSIONThe cells infected with L.interrogans of different virulence show distinct intracellular free Ca(2+)levels and peak patterns. The different host cell lines can affect the intracellular free Ca(2+)levels, which is not related to the PLC activity in the leptospiral strains.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Cercopithecus aethiops ; Endocytosis ; Humans ; Leptospira interrogans ; enzymology ; pathogenicity ; Macrophages ; metabolism ; microbiology ; Type C Phospholipases ; metabolism ; Vero Cells ; Virulence

OBJECTIVETo explore the possibility of repairing periodontal defects with carbonated calcium phosphate bone cement (CCPBC) modified with synthesized peptides.

METHODSPeriodontal bone defects in 4 dogs were surgically created and then restored directly with hydroxyapatite (HA), Perioglass, CCPBC and CCPBC modified with peptides. The results were compared at different levels.

RESULTSBone replacement materials were lost in HA and Perioglass groups. In the HA group defects were restored with connective tissue. Perioglass group had only a little new bone around materials by alveolar bone. CCPBC could firmly stay in bone defects to maintain the space of bone defects even without membrane use. CCPBC modified with peptides was superior to HA, Perioglass, and CCPBC, surrounded by a great deal of new bone.

CONCLUSIONUnder limitation of this study, CCPBC modified with peptides has some osteoinuctive activity and may have good prospect for the clinical application in periodontal defect repair.

Alveolar Bone Loss ; therapy ; Animals ; Bone Cements ; Bone Regeneration ; Bone Substitutes ; Calcium Phosphates ; Dogs ; Durapatite ; Male

OBJECTIVETo establish an improved procedure for isolation and identification of epitopes from a random peptide library displayed on the bacterial surface.

METHODSEpitopes were screened from FliTrx random peptide library by a monoclonal antibody 3B9 against HBV-PreS2 protein. The enrichment was monitored in each round. Higher affinity clones were obtained by increasing the washing strength and randomly selected for sequencing and Western blot analysis.

RESULTSClones specifically binding to antibody were enriched in each round. Ten sequences were obtained from sixteen sequenced clones, seven of them contained the common motif RXRGXY with high homogeneity to 135-140 amio acids in HBV-PreS protein and have positive results in Western blot analysis. The other three sequences have no typical motif RXRGXY and showed different Western blot results.

CONCLUSIONSIt's easy and quick to drive epitopes from a random peptide library displayed on the bacterial surface.

Amino Acid Sequence ; Animals ; Antigens, Viral ; immunology ; Bacterial Proteins ; immunology ; Flagellin ; immunology ; Hepatitis B Surface Antigens ; genetics ; immunology ; isolation & purification ; Immunodominant Epitopes ; immunology ; Molecular Sequence Data ; Peptide Library ; Protein Precursors ; genetics ; immunology ; isolation & purification

Small cell carcinoma of the esophagus (SCCE) is a rare and aggressive malignant tumor with a poor prognosis. The optimal disease staging system and treatment approaches have not yet been defined. This study aimed to evaluate the prediction of different staging systems for prognosis and treatment options of SCCE. We retrospectively accessed the clinicopathologic characteristics, treatment strategy, and prognosis of 76 patients diagnosed with primary SCCE between 2001 and 2011. The 1-, 2-, 3-, and 5-year overall survival rates were 58%, 31%, 19%, and 13%, respectively. Univariate analysis showed that the 2002 American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) classification (P = 0.002), Veterans Administration Lung Study Group (VALSG) stage (P = 0.001), predisposing factors (P < 0.001), T category (P = 0.023), and M category (P < 0.001) were prognostic factors for overall survival. Multivariate analysis showed that the 2002 AJCC TNM stage (P < 0.001) was the only independent prognostic factor for survival. The value of the area under the receiver operator characteristic (ROC) curve (AUC) of the 2002 AJCC TNM staging system was larger than that of VALSG staging system with regard to predicting overall survival (0.774 vs. 0.620). None of the single treatment regimens showed any benefit for survival by Cox regression analysis. Thus, the 2002 AJCC TMN staging system improved the prediction of SCCE prognosis; however, the optimal treatment regimen for SCCE remains unclear.
Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma, Small Cell ; classification ; pathology ; therapy ; Cisplatin ; administration & dosage ; Combined Modality Therapy ; Esophageal Neoplasms ; classification ; pathology ; therapy ; Esophagectomy ; methods ; Etoposide ; administration & dosage ; Female ; Humans ; Lymph Node Excision ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; methods ; Paclitaxel ; administration & dosage ; Radiotherapy, High-Energy ; Retrospective Studies ; Societies, Medical ; Survival Rate ; United States

OBJECTIVETo study the immunogenicity and immunoprotection of the recombinant expressing product (rSpaO) of S. paratyphi A spaO gene, and to demonstrate the frequencies of spaO gene carrying and expressing in S. paratyphi A isolates.

METHODSThe spaO gene of a clinical S. paratyphi A strain JH01 was amplified and then cloned. After sequencing of the cloned spaO gene, a prokaryotic expression system of the gene was constructed. SDS-PAGE were applied to examine the rSpaO expression. Ni-NTA affinity chromatography was performed to collect rSpaO. Immunogenicity of rSpaO was determined by Western blot assay. A PCR assay and an ELISA were established to respectively detect the carrying and expressing frequencies of the spaO genes in 98 S. paratyphi A isolates. The immunoprotective effects of rSpaO in S. paratyphi A strain 50001 infected mice were observed.

RESULTSIn comparison with the reported corresponding sequences, the nucleotide and putative amino acid sequence homologies of the cloned spaO gene were 99.45%-99.89% and 99.01%-100%, respectively. The expression output of rSpaO was approximately 75% of the total bacterial proteins. S. paratyphi A antiserum could recognize as well as combine with rSpaO. rSpaO could efficiently induce rabbits to produce specific antibody. 94.9% (93/98) of the S. paratyphi A isolates had spaO gene and 91.4% (85/93) of the spaO+ strains could express SpaO. 58.3% and 50.0% of the mice that oral-taken or subcutaneous injected with 500 microg of rSpaO for immunization were survival after challenged by lethal dose of S. paratyphi A strain 50001. When co-immunized with 5 microg rLTB, the survival rates of the mice increased to 88.3% and 75.0%, respectively.

CONCLUSIONThe S. paratyphi isolates had relatively high carrying and expressing frequencies of spaO gene. rSpaO showed a fine immunogenicity and a certain immunoprotective effect, which could be used as an antigen candidate for developing genetic engineering vaccine of S. paratyphi.

Animals ; Antibody Formation ; Bacterial Proteins ; genetics ; immunology ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Bacterial ; Genetic Engineering ; Membrane Proteins ; genetics ; immunology ; Mice ; Polymerase Chain Reaction ; Recombinant Proteins ; Salmonella Vaccines ; immunology ; Salmonella paratyphi A ; genetics

Small intestinal obstruction is a common complication of primary gastrointestinal cancer or metastatic cancers.Patients with this condition are often poor candidates for surgical bypasses,and placement of self-expanding metal stent (SEMS) can be technically challenging.In this study,we examined the feasibility of combined application of single-balloon enteroscope (SBE) and colonoscope for SEMS placement in patients with malignant small intestinal obstruction.Thirty-four patients were enrolled in this study,among which 22 patients received SEMS placement by using SBE and colonoscope,while the other 12 patients received conservative medical treatment.The patients were followed up for one year.Stent placernent was technically feasible in 95.5％ (21/22).Clinical improvement was achieved in 86.4％ (19/22).For the 19 clinical success cases,the average time of benefits from a gastric outlet obstruction scoring system (GOOSS) increase ≥1 was 111.9±89.5 days.For the 12 patients receiving conservative medical treatment,no significant improvement in GOOSS score was observed.Moreover,a significant increase of Short-Form-36 health survey score was observed in the 19 patients at time of 30 days after stent placement.By Kaplan-Meier analysis,a significant survival improvement was observed in patients with successful SEMS placement,compared with patients receiving conservative medical treatment.Taken together,combined use of SBE and colonoscope makes endoscopic stent placement feasible in patients with malignant small intestinal obstruction,and patients can benefit from it in terms of prolonged survival and improved quality of life.

OBJECTIVETo construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients.

METHODSlipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method.

RESULTSThe homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable.

CONCLUSIONlipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.

Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; immunology ; Bacterial Outer Membrane Proteins ; genetics ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Bacterial ; Genes, Bacterial ; genetics ; Humans ; Leptospira interrogans ; genetics ; Leptospirosis ; immunology ; microbiology ; Lipoproteins ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology