ABSTRACT
Objective:To investigate the expression of Per2 mRNA, HDAC1 mRNA and E-cadherin mRNA in esophageal cancer cells and their significance.Methods:The experimental study was conducted. Human normal esophageal epithelial cells as the control group and human esophageal cancer cell line KYSE-150 cells as the experimental group were cultured in vitro to logarithmic growth stage. Observation indicators: (1) the proliferation of cells; (2) the migration and invasion of cells; (3) the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA in cells of initial physiological state; (4) the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA after cells were treated with Per2-agonists or inhibitors; (5) the expression of Per2 mRNA and E-cadherin mRNA after cells were treated with HDAC1 inhibitors. Measurement data with normal distribution were represented as Mean± SD, the t test was used for comparison within groups and the t test or ANCOVA were used for comparison between groups. Results:(1) The proliferation of cells: the cell proliferation of the experimental group and control group were 0.90%±0.14% and 0.52%±0.08%, with a significant difference between the two groups ( t=5.166, P<0.05). (2) The migration and invasion of cells: the numbers of cell migration and invasion for the experimental group were 173±41 and 86±27, versus 50±15 and 21±9 for the control group, with significant differences between the two groups ( t=6.274, 5.153, P<0.05). (3) The expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA in cells of initial physiological state: the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA in cells of initial physiological state for the experimental group was 11.7±2.7, 20.4±6.6, and 12.4±2.5, respectively, versus 2.4±0.5, 8.5±2.2, and 27.3±4.5 for the control group, with significant differences between the two groups ( t=5.782, 2.982, -5.034, P<0.05). (4) The expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA after cells were treated with Per2-agonists or inhibitors: after cells were treated with Per2-agonists, the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA were 13.1±2.2, 22.4±6.2, 16.6±4.2 for the experimental group, and 9.9±3.1, 18.4±5.6, 15.3±2.3 for the control group, respectively. There was no significant difference in the expression of Per2 mRNA, HDAC1 mRNA, or E-cadherin mRNA of the experimental group between cells being treated with and without Per2-agonists ( t=-4.300, 10.087, -4.187, P>0.05). There were significant differences in the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA of the control group between cells being treated with and without Per2-agonists ( t=-4.846, 3.501, 9.294, P<0.05). There was no significant difference in the expression of Per2 mRNA or E-cadherin mRNA between the experimental group and control group after cells were treated with Per2-agonists ( F=1.000, 7.582, P>0.05), while there was a significant difference in the expression of HDAC1 mRNA between the two groups ( F=1.724, P<0.05). After cells were treated with Per2-inhibitors, the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA were 4.1±1.7, 7.5±2.2, 22.8±4.2 for the experimental group, and 3.1±0.9, 9.3±3.2, 28.4±5.8 for the control group, respectively. There were significant differences in the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA of the experimental group between cells being treated with and without Per2-inhibitors ( t=12.124, 5.105, -10.245, P<0.05). There was no significant difference in the expression of Per2 mRNA, HDAC1 mRNA, or E-cadherin mRNA of the control group between cells being treated with and without Per2-inhibitors ( t=-2.815, 1.568, -1.439, P>0.05). There were significant differences in the expression of Per2 mRNA and E-cadherin mRNA after cells were treated with Per2-inhibitors between the experimental group and control group ( F=22.965, 82.134, P<0.05), while there was no significant difference in the expressions of HDAC1 mRNA between the two groups ( F=6.416, P>0.05). (5) The expression of Per2 mRNA and E-cadherin mRNA after cells were treated with HDAC1 inhibitors: after cells were treated with HDAC1 inhibitors, the expression of Per2 mRNA and E-cadherin mRNA were 13.4±3.5, 24.2±3.4 for the experimental group, and 3.1±1.2, 26.8±5.2 for the control group, respectively. There was no significant difference in the expression of Per2 mRNA of the experimental group between cells being treated with and without HDAC1-inhibitors ( t=-3.959, P>0.05). There was a significant difference in the expression of E-cadherin mRNA of the experimental group between cells being treated with and without HDAC1-inhibitors ( t=-21.977, P<0.05). There was no significant difference in the expression of Per2 mRNA or E-cadherin mRNA of the control group between cells being treated with and without HDAC1-inhibitors ( t=-1.440, 1.058, P>0.05). After cells were treated with HDAC1-inhibitors, there was no significant difference in the expressions of Per2 mRNA between the experimental group and control group ( F=2.004, P>0.05), while there was a significant difference in the expression of E-cadherin mRNA between the two groups ( F=325.800, P<0.05). Conclusions:Human esophageal cancer cells have an elevated expression of Per2 mRNA and HDAC1 mRNA, and a reduced expression of E-cadherin mRNA. The overexpression of Per2 mRNA may activate the expression of downstream targeting protein HDAC1, and inhibit the expression of cell surface E-cadherin mRNA.
ABSTRACT
Objective To evaluate the characteristics of spatial distribution and time accumulation of impact acceleration at different parts of human body during backward falling process. Methods Four healthy men and four healthy women (20-20 years old) were enrolled. The tri-axial acceleration on head, chest, left/right arm/hand/foot, left/right front/back hip, left/right femur head, sacrum and coccyx throughout the backward falling were measured by ADXL335 tri-axial acceleration sensor. Systemic acceleration distribution of backward falling was polynomial fitted by signal magnitude vector (SMV) of its first peak. Besides, parameters of impulse mechanics such as zero-g time, total falling time, peak SMV, relative pressure impulse of the vulnerable sites (head, hip and its related sites) were also calculated. Results Compared with the other parts of the body, the peak SMV and relative impulses of left/right back hip and head were significantly higher (P＜0.05). Acceleration that paralleled to the ground in left/right back hip was also relatively large. The rotational transform angles of left/right back hip, left/right femur head, sacrum and coccyx were significantly larger (P＜0.05). In addition, during the process of falling backward to the ground, a sliding tendency toward the sagittal plane 53.58°±6.75° occurred at all testing sites. Conclusions Head and hips are vulnerable during backward falling, and their zero-g time (0.26±0.05) s can be used as the longest starting time of falling protection devices. The large change angle of left/right hip, left/right femoral head, sacrum and coccyx may be the important cause of the sprain during backward falling.
ABSTRACT
Objective To explore the carry rate and antimicrobial resistance of Staphylococcus aureus (S.aureus) among primary school students.Methods Nasal swab samples were collected from healthy primary school students in Guangzhou.Antibiotic susceptibility testing was applied to test S.aureus strains.Results The overall carriage rate of S.aureus,methicillin-resistant S.aureus (MRSA) and multi-drug-resistant S.aureus (MDRSA) among 1 012 primary school students were 40.1％,1.2％ and 4.0％,respectively.Most S.aureus isolates were resistant to penicillin,erythromycin and clindamycin.The dominant multidrug resistance patterns of MDRSA isolates were resistant to erythromycin-clindamycin-tetracycline and erythromycin-clindamycin-cefoxitin.Multifactor dimensionality reduction analysis showed that the rate of resistance to cefoxitin,tetracycline and chloromycetin among MDRSA was 104.39 times as much as that of nonMDRSA.Conclusions The carriage rate of S.aureus in healthy primary school students from Guangzhou was high and these isolates showed multidrug resistance.These data provide basis for guiding the rational use of antibiotics.
ABSTRACT
Objective To explore the role of IL-1β in capillary leak syndrome by observing the alterations of AQP-1 expression,apoptosis,and ultrastructural of vascular endothelial cells under the action of IL-1β.Methods Umbilical vein endothelial cells (UVEC) in vitro were randomly allocated into 3 groups:time,concentration,and control.In the time group,UVECs were treated with culture medium containing 20 μg/L IL-1β for3 h(T1),8 h(T2),12 h(T3) and 24 h(T4).In the concentration group,UVECs were treated with culture medium containing 0.2 μg/L(C1),2 μg/L(C2) and 20 μg/L(C3) IL-1β for 24 h.In the control group,UVECs were treated with culture medium without IL-1β for 24 h.The changes of AQP-1 mRNA and protein expression were detected by real-time PCR and Western blot.Apoptosis was detected by flow cytometry,and cell ultrastructural changes were observed by electron microscopy.Results AQP-1 mRNA and protein expression of T1-T4 in the time group and C1-C3 in the concentration group were lower than those of the control group (P ＜ 0.05).The apoptotic rate was increased,and mitochondrial swelling,vacuolar degeneration,karyolysis and necrosis were observed under electron microscopy.These were more pronounced with time or concentration increases.Conclusions IL-1β can cause a decrease of AQP-1 mRNA and protein expression,increase in apoptotic rate and increase in damage to the cells'ultrastructure.This is an important reason for damage to the vascular endothelial barrier and may be associated with capillary leak syndrome.
ABSTRACT
Objective To investigate the mechanism of cyclic adenosine monophosphate / protein kinase A signal transduction pathway in severe acute pancreatitis(SAP)-associated lung injury.Methods Seventy-two male healthy SD rats were completely randomized into three groups:sham operation (SO) group(n =8),SAP group and SAP plus H89 (cAMP inhibitor) group,then the latter two groups were divided into four sub-groups with eight rats in each sub-group according to the sampling time of 3,6,12 and 24 h,and the total numbers of groups were nine.The content change of TNF-α and IL-1β in serum,protein levels of cAMP-dependent protein kinase catalytic subunit (PKA C) and phosphorylated vasodilator-stimulated phosphoprotein(p-VASP) and the expression of VSAP mRNA in lung tissue were detected by enzyme-linked immunosorbent assay (ELISA),immunohistochemistry and quantitative real time PCR,respectively.Pathological changes of the pancreas and lung tissues were also observed.Results Compared with the SO group,the serum levels of TNF-α and IL-1β in the SAP group were obviously increased at different time points(P ＜0.05).Pathological changes of the pancreas and lung tissues were aggravated significantly.The protein levels of PKA C,p-VASP and the expression of VSAP mRNA in lung tissue were increased significantly (P ＜0.05)which peaked at 12 h in the SAP group [TNF-α was (266.07 ± 17.14) pg/mL,IL-1β(169.17 ±25.92) pg/mL,PKA C(210.69 ±6.32) × 103,p-VASP (56.62 ±0.57) × 103,VASP mRNA(2.06 ±0.21)],which had positive correlation with the serum level of TNF-α and IL-1β.Compared with the SAP group,pathological changes of the pancreas and lung tissues were alleviated significantly,the protein levels of PKA C,p-VASP and the expression of VSAP mRNA in lung tissue were decreased significantly in the SAP plus H89 group at different time points(P ＜ 0.05).Conclusion The cyclic adenosine monophosphate / protein kinase A signal transduction pathway is found to participate in the pathological process of SAP-associated lung injury through the up-regulations of TNF-α,IL-1 β and phospho-VASP.
ABSTRACT
Objective To investigate the expression and significance of E-selectin in lung tissue of rat of acute necrotizing pancreatitis (ANP)-induced lung injury.Methods Rats were randomly divided into control and ANP groups.ANP model was induced by retrograde injection of sodium taurocholate into the biliary and pancreatic duct.The rats were sacrificed at 3,6,12 and 24 h.Serum amylase,albumin,Ca2+ and lung wet/dry (W/D) weight ratio were determined,and pancreas and lung tissues underwent routine pathological examination and histopathological scores were evaluated.The expression of E-selectin mRNA and protein in lung tissue of rat was measured by real-time fluorescence quantitative RT-PCR and immunohistochemistry.Results In ANP group at 6h,the serum level of amylase,histopathological scores of pancreas and lung tissues,lung W/D weight ratio were (3978 ± 476) U/L,8.22 ± 0.63,( 12.31 ± 1.58,5.21 ± 0.20,which were significantly higher than those in control group [ (843 ± 90)U/L,0.22 ± 0.36,0.13 ± 0.34,4.46 ±0.17,P ＜ 0.05 or ＜ 0.01 ],the serum level of albumin was (12.9 ± 1.0)g/L,which was significantly lower than that in control group [ ( 15.6 ± 0.7 ) g/L,P ＜ 0.01 ],the serum level of Ca2 + at 12 h was ( 2.33 ±0.15) mmoL/L,which was significantly lower than that in control group [(2.57 ± 0.23)mmoL/L,P ＜ 0.01 ].E-selectin was located in the cell membrane and cytoplasm of vascular endothelial cells.Expression of E-seleetin mRuNA and protein in the lung tissue at 6h was significantly higher than those in control group (3.51 ±0.45 vs 0.95 ± 0.16,0.174 ± 0.019 vs 0.060 ± 006,P ＜ 0.01 ).Conclusions Pulmonary expression of E-selectin of vascular endothelial cell is significantly up-regulated in a time dependant manner when ANPinduced ALI occurs; and it is involved in leukocyte mural and extravasation,and can promote lung injury.
ABSTRACT
We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (T(m)) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the T(m) of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the T(m) difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.
Subject(s)
DNA-Binding Proteins , Genetics , Diphosphates , Metabolism , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Recombinant Proteins , Genetics , Sequence Analysis, DNA , MethodsABSTRACT
Pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate (PEP) to ATP, Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp. aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET28a(+), yielding pET28a (+)-PPDK. The E. coli BL21 (DE3) was transformed with the pET28a (+)-PPDK. After inducing with IPTG, the E. coli BL21 (DE3) [pET28a (+)-PPDK] expressed recombinant PPDK fused to an N-terminal sequence of 6-His Tag. The molecular weight of PPDK was estimated to be 101 kD by SDS-PAGE. The PPDK was purified by His * Bind Resin affinity chromatography and ultrafiltration using 10 kD cut-off membrane. The successful application of PPDK in pyrosequencing was also demonstrated.