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 investigate weather there is a toll-like receptor 4 (TLR4)-mediated myeloid differentiation factor 88 (MyD88)-dependent pathway in hippocampal neurons of rats and the probable role of the pathway in neuroinflammation.

METHODSTo establish the proper model, primarily cultured hippocampal neurons were treated with lipopolysaccharides (LPS), or pretreated with TLR4 antibody then co-treated with LPS. The expression of mRNA of MyD88 and TNF-alpha receptor associated factor 6 (TRAF6) were tested by RT-qPCR. The content of MyD88 and TRAF6 were tested by Western blot. The nuclear translocation of nuclear factor-kappaB/P65 (NF-kappaB/p65) was tested by immunofluorescence. The content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and nitric oxide (NO) were tested by ELISA.

RESULTSLPS could increase MyD88 and TRAF6 mRNA, upregulate protein level of MyD88 and TRAF6 and increase the level of TNF-alpha, IL-1beta and NO in cell culture supernatant. LPS also could promote NF-kappa B/p65 translation to the nucleus. The pretreatment with TLR4 antibody reduced the translocation to nucleus for NF-kappaB/P65 and the contents of TNF-alpha, IL-1beta and NO in the culture supernatant.

CONCLUSIONThere is a TLR4-mediated MyD88-dependent pathway in hippocampal neurons. The activation of this pathway can increase the level of TNF-alpha, IL-1beta and NO in cell culture supernatant. TLR4-mediated MyD88-dependent pathway in hippocampal neurons participate in neuroinflammation, that means neurons are not passive in inflammation.

Animals ; Cells, Cultured ; Hippocampus ; cytology ; metabolism ; Interleukin-1beta ; metabolism ; Myeloid Differentiation Factor 88 ; metabolism ; Neuritis ; metabolism ; Neurons ; metabolism ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; TNF Receptor-Associated Factor 6 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo explore a new technique for the treatment of lower lip defect after carcinoectomy.

METHODSix lower lip defects (more than two third of the length of the lower lip) after the tumor resection were treated with an orbicular muscular-mucous advancement flap.

RESULTSAll of the patients had achieved good results functionally and cosmetically with the following-ups from 6 months to 3 years.

CONCLUSIONThe above mentioned techique could be a simple, safe and effective method for repairing lower lip defect.

Adult ; Aged ; Carcinoma, Squamous Cell ; surgery ; Female ; Humans ; Lip ; pathology ; surgery ; Lip Neoplasms ; surgery ; Male ; Middle Aged ; Postoperative Complications ; surgery ; Reconstructive Surgical Procedures ; methods ; Surgical Flaps

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

OBJECTIVETo investigate the immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans.

METHODSNi-NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2, LipL32/1 and rLipL32/2, LipL41/1 and rLipL41/2 expressed by the different genotypes. By using Signal P-NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class-II binding peptide prediction-ProPred prediction server, the possible signal peptides, MHC-II binding peptides and lymphocyte B epitopes were analyzed. The IL-1, IL-8 and TNF-alpha secretion in human umbilical vein endothelial cell line EVC-304 induced by target recombinant proteins were measured by ELISA.

RESULTSUnder the inducement of IPTG, the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%, 40% and 35%, and 15% and 10% of the total bacterial proteins, respectively. Each of the purified target recombinant proteins showed a single protein band in SDS-PAGE. The signal peptides of OmpL1s, LipL32/1 and LipL32/2, and LipL41s were located at the N ends of 1-24, 1-21 and 1-24, and 1-24 amino acid residuals, respectively. OmpL1s, LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC-II binding peptides and lymphocyte B and OmpL1/2 had another one (59-78). The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL-1alpha , IL-8 and TNF-alpha (P<0.05) in EVC-304 cells. The IL-1alpha levels reached the highest at the 24 h and then declined,while the IL-8 and TNF-alpha levels after 48 h treatment were higher that those after 24 h.

CONCLUSIONThe expression products in ompL1/1, lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes. rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC-304 cells.

Bacterial Outer Membrane Proteins ; immunology ; pharmacology ; Cells, Cultured ; Endothelial Cells ; cytology ; Epitopes ; Genotype ; Humans ; Inflammation ; etiology ; Interleukin-1 ; biosynthesis ; Leptospira interrogans ; genetics ; immunology ; Lipoproteins ; immunology ; pharmacology ; Recombinant Proteins ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis ; Umbilical Veins ; cytology

OBJECTIVETo determine the ability of adhering and invading to host cells and the related pathologic changes of Leptospira spp. with different virulence.

METHODSA special Fontana silver staining method was developed to observe the ability of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and serogroup Pomona serovar pomona strain 56608, and L.biflexa serogroup Samaranga serovar patoc strain Patoc I to adhere Vero and J774A.1 cells. Ultrastructural lesions of the infected cells were examined by electron microscopy. By using flow cytometry with fluorescein labeling of FITC-Annexin V/PI,apoptosis and necrosis of the Vero and J774A.1 cells induced by the leptospiral strains before and after inactivation with ultraviolet treatment were detected, respectively.

RESULTSThe adhering rates of L.interrogans strains 56601 and 56608 to the Vero cells were 24.2% and 22.9% (P>0.05), while to the J774A.1 cells were 49.0% and 46.9% (P>0.05), respectively. L.biflexa strain Patoc I did not adhere to these two host cells. After the two strains of L.interrogans invaded two different cell lines, the special phagosomes containing the Leptospira were formed and similar cell ultrastructural lesions, such as chromatilysis and chromatin condensation, vacuolar degeneration, mitochondrion swelling and mitochondrial crista disappearance, and endoplasmic reticulum swelling and paramembranous ribosome disappearance, were observed. The apoptosis rates in the Vero cells caused by the L.interrogans strains 56601 and 56608 before and after ultraviolet inactivation were 84.4%, 82.8% and 77.9%, 86.1%, respectively. The L.interrogans strain 56601 mainly induced the terminal apoptosis in Vero cells with the rates of 68.0% and 52.9% before or after inactivation, while the L.interrogans strain 56608 mainly induced the early apoptosis in Vero cells with apoptosis rates of 64.1% and 50.1% before or after inactivation. In the J774A.1 cells, the L.interrogans strain 56608 caused cell necrosis (before and after inactivation) and apoptosis that was dominated by terminal apoptosis.

CONCLUSIONThe established Fontana silver staining method can be used to observe adhesion of L.interrogans.L.interrogans can invade host cells through endocytosis which causes untrastructural lesions. The necrosis or apoptosis induced by L.interrogans are affected by different host cell lines but not the virulence of L.interrogans strains.

Animals ; Apoptosis ; physiology ; Cell Adhesion ; Cells, Cultured ; Cercopithecus aethiops ; Endocytosis ; Humans ; Leptospira interrogans ; classification ; pathogenicity ; Macrophages ; microbiology ; ultrastructure ; Serotyping ; Vero Cells ; Virulence

OBJECTIVETo construct prokaryotic expression systems of ltB/ctB-lipL41/1 fusion genes, identify immunogenic and adjuvant activities of the products as well as to understand the frequencies of lipL41 gene that carrying and expressing in L. interrogans wild strains and specific antibody levels in sera from patients with leptospirosis.

METHODSPolymerase chain reaction (PCR) with linking primer was applied to construct the fusion genes ltB-lipL41/1 and ctB-lipL41/1. By routine molecular biological techniques, prokaryotic expression systems of the two fusion genes were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine expression of the target recombinant proteins rLTB-rLipL41/1 and rCTB-rLipL41/1. Both western blot and Ganglioside-enzyme linked immunosorbent assay (GM-ELISA) were used while immunogenic and adjuvant activities of rLTB-rLipL41/1 and rCTB-rLipL41/1 were measured. PCR and MAT were performed to detect lipL41 gene and expression of the gene in 97 wild strains of L. interrogans, respectively. Antibodies against product of lipL41 gene in serum samples from 228 leptospirosis patients were detected by ELISA.

RESULTSIn comparison with reported corresponding sequences, the similarities of ltB-lipL41/1 and ctB-lipL41/1 fusion genes to nucleotide and putative amino acid sequence were 99.6%-99.9% and 99.8%-100%, respectively. Expression outputs of both rLTB-rLipL41/1 and rCTB-rLipL41/1, mainly presenting with inclusion body, consisting approximate 10% of the total bacterial proteins. Both rLTB-rLipL41/1 and rCTB-rLipL41/1 could combine rabbit anti-rLipL41/1 serum as well as bovine GM1, respectively. 87.6% of the L. interrogans wild strains(85/97) having lipL41 gene while 84.5% (82/97) of the wild strains with rLipL41/1 or rLipL41/2 antiserum were positive for MAT with titers of 1:4-1:128. 84.6% (193/ 228), 78.5% (179/228) from the patients' serum samples were positive for rLipL41/1 and rLipL41/2 antibodies, respectively.

CONCLUSIONltB-lipL41/1 and ctB-lipL41/1 fusion genes and their prokaryotic expression systems were successfully constructed in this study. The two expressed fusion proteins showed qualified immunogenic and adjuvant activities. lipL41 gene was extensively distributed and frequently expressed in different serogroups of L. interrogans. rLTB-rLipL41/1 or rCTB-rLipL41/1 seemed to have had good potential to serve as an antigen in L. interrogans genus-specific vaccine.

Amino Acid Sequence ; Animals ; Antibody Specificity ; Antigens, Bacterial ; biosynthesis ; chemistry ; genetics ; immunology ; Cattle ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli Proteins ; genetics ; Genetic Engineering ; methods ; Humans ; Leptospira interrogans ; genetics ; physiology ; Leptospirosis ; immunology ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; immunology ; Sequence Analysis, DNA