Adult ; Altitude ; Arteries ; metabolism ; Calcitonin Gene-Related Peptide ; blood ; Humans ; beta-Endorphin ; blood

Objective To establish a CLCN3/MMTV-PyMT double transgenic mouse model of spontaneous breast tumor with simultaneously overexpressing ClC-3.Method CLCN3 transgenic mice were crossed with MMTV-PyMT spon-taneous mammary tumor model mice.The genotype was determined by PCR.The expression of ClC-3 in tissues was detec-ted by immunofluorescence and Western blot.Results CLCN3 and MMTV-PyMT transgenic mice were bred and CLCN3/MMTV-PyMT hybrid mouse model was successfully established.The ClC-3 expression in CLCN3/MMTV-PyMT hybrid mice was higher than that in the MMTV-PyMT mice, assessed by immunofluorescence and Western blot analysis.Conclu-sions Transgenic mouse models of spontaneous breast cancer with simultaneously overexpressing ClC-3 are successfully es-tablished.The double transgenic mice provide a good animal model for further research of ClC-3 in tumor growth and metas-tasis.

Objective To explore the influence of fluorine on mRNA and protein expression of the insulin-like growth factor-1 (IGF-1) and its receptor of rat osteoblasts.Methods Osteoblasts were isolated from rat bone by enzyme digestion.Different fluorine concentration [0 (control),10-7,10-6,10-5,10-4,10-3 mol/L] was add to the second generation osteoblasts.The IGF-1 in the culture medium was determined by radioimmunoassay (RIA) at different fluorine concentration and different time (24,48 h).The expression of IGF-1 receptor was measured by the method of fluorescent quantitation PCR and the expression of protein IGF-1 receptor was measured by Western blotting.Results ①With increased dose of fluoride exposure,IGF-1 concentration in the osteoblastic culture medium increased first and then decreased at 24,48 h,respectively.Compared to the control group [(38.83 ± 3.48)ng/L],IGF-1 concentration of the 24 h 10-6 mol/L group[(65.45 ± 4.84)ng/L] was higher,and the difference was statistically significant(P ＜ 0.05).The same result was also shown in the 48 h 10-5 mol/L group [(59.14 ± 1.53)ng/L] to its corresponding control group [(33.79 ± 1.84)ng/L,P ＜ 0.05].②The mRNA expression of IGF-1 receptor of the 24,48 h 10-5 mol/L groups (0.0055 ± 0.0004,0.0262 ± 0.0040) was significantly higher than their corresponding control groups (0.0022 ± 0.0001,0.0073 ± 0.0008,all P ＜ 0.05).③With increased dose of fluoride exposure,the protein expression of IGF-1 receptor increased first and then decreased ;the expression of 24 h 10-5 mol/L group (1.39 ± 0.16) was compared with the corresponding control group (0.86 ±0.12),and the difference was statistically significant (P ＜ 0.05) ; the expression of 48 h every fluorine group was also compared with the corresponding control group,and the difference was not statistically significant(all P＞ 0.05).Conclusions Fluorine can affect the mRNA and protein expression of osteoblastic IGF-1 and its receptor.It indicates that IGFS signal transduction pathways play an important role in fluorine regulation of bone metabolism.

Aim To establish a model of the vascular endothelial cell (VEC) injury in SDrats.Methods SD rats were randomly divided into the control and the modelgroups. The model rats were injected with adrenaline diluted to 2. 5 times 0. 05 mg?100 g-1 (tid) for 5 d continously. From the 4th d, they were irritated for 5 min in the0℃ cold-water in the middle between adrenaline injections.The control rats weregiven 0. 9% NS as above. At 6th d, blood samples were taken from carotid arteries ofthe rats and the CEC counts, t - PA、PAI activities, 6-keto-PGF1? concentrations andthe platelet aggregation rate(max) were detected respectively. Results In the modelgroup, as compared with those in the control group, t - PA activity and 6-keto-PGF1?concentration decreased significantly(P

Objective To investigate correlation between aspirin resistance(AR) and inflammatory factors. Methods One hundred and ten patients with coronary heart disease took aspirin 0.1 mg/d for 14 days.It was detected platelet aggregation function induced with adenosine disphosphate (ADP) and arachidonic acid (AA), and investigated correlation between AR and inflammatory factors. Interleukin-1? (IL-1?),interleukin-6 (IL-6) and high sensitive C-reaction protein (hs-CRP) levels. Results IL-6 level of patients with AR was significantly higher than that of aspirin sensitive (AS) patients. The other two index were not different between the two groups. Conclusion IL-6 levels could be used as predictor.

Objective To investigate whether the effect of puerarin on right ventricle hypertrophy of pulmonary hypertensive rats induced by chronic hypoxia-hypercapnia was related to new peptide Apelin or its receptor(APJ).Methods Thirty male Sprague-Dawley rats were randomly divided into three groups, they are control group,hypoxia-hypercapnia 4-week model group,and hypoxia-hypercapnia 4-week plus puerarin group.The concentrations of Apelin-36 protein in plasma and homogenate of right ventricular muscle were detected by radioimmunoassay.The mRNA expressions of Apelin and APJ in right ventricu- lar muscle were measured by semi-quantitive reverse transcription polymerase chain reaction(RT-PCR). Results The weight ratio of right ventricle to left ventricle plus septum[RV/(LV+S)] in model group was significantly higher than that in control group(P0.05).The plasma concen- tration of Apelin-36 protein in model group was significantly higher than that in control group(P

OBJECTIVETo investigate serum level and gene polymorphisms of matrix metalloproteinase 9 (MMP-9), and platelet glycoprotein VI (GPVI) in patients with acute coronary syndrome (ACS).

METHODSIn a prospective study of 179 patients with documented ACS and 164 controls, we measured baseline serum MMP-9 levels using ELISA and determined the MMP-9/C-1562T and MMP-9/G5564A genotypes using PCR-restriction fragment length polymorphism. Fib serum level was measured by Clauss assay. We also analyzed the Fib/Bbeta-148C/T and GPVI/T13254C polymorphisms.

RESULTSSerum levels of MMP-9 and Fib in ACS patients were significantly higher than in controls (P < 0.001), and serum level of Fib in the acute myocardial infarction group was higher than in patients with unstable angina (P < 0.05). No significant difference between ACS patients and controls was found in frequencies of MMP-9/C-1562T, MMP-9/G5564A, Fib/Bbeta-148C/T, and GPVI/T13254C genotypes and alleles (P > 0.05). The T allele of the Fib/Bbeta-148T polymorphism was associated with increased plasma Fib level (P < 0.05). There was a strong positive correlation between serum level of MMP-9 and Fib (r = 0.289, P < 0.01).

CONCLUSIONSerum levels of MMP-9 and Fib were independent risk factors of ACS. There was an obvious relationship between the Bbeta-148C/T mutation and high Fib level. No significant difference between controls and ACS patients was found in the frequencies of MMP-9 C-1562T and G5564A, Fib Bbeta-148C/T and GPVI T13254C genotypes and alleles (P > 0.05).

Acute Coronary Syndrome ; genetics ; Adult ; Aged ; Case-Control Studies ; Female ; Humans ; Male ; Matrix Metalloproteinase 9 ; blood ; genetics ; Middle Aged ; Platelet Membrane Glycoproteins ; genetics ; Polymorphism, Single Nucleotide

Objective To establish a convenient,accurate and practical method for detection of adefovir dipivoxil resistance-as- sociated mutation in hepatitis B virus:rtA181V/T/S and rtN236T mutations.Methods According to HBV complete sequences in GenBank,two pairs of primers were designed to amplify the region of HBV reverse transcriptase in order to introduce a BglI restriction site upon PCR product of wild type (wt) and a BseDI restriction site upon PCR product of rt236 mutant type.After amplification,the PCR products were digested with BglI and BseDI separately.We used this method to detect wild,rt181 mu- tant,rt236 mutant plasmids and 3 chronic hepatitis B patients' serum with obvious ADV resistance-associated mutations.We also tested the sensitivity of this method by mixing the wild and mutant plasmids in different proportions.Results The method could detect rt181 and rt236 mutations simultaneously.The result of RFLP analysis was in accordance with that of DNA se- quencing and cloning analysis.This method could detect the mutants even when they comprised only 10% of the total virus population.Conclusions The PCR-RFLP method with high sensitivity can detect rt181 and rt236 mutations simultaneously.It can be used for early detection of ADV resistance-associated mutation in hepatitis B virus.

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

OBJECTIVETo investigate the anti-angiogenic effect of cryptotanshinone (CPT) on human umbilical vein endothelial cells (HUVECs) and the effect of CPT on Wnt/β-catenin signaling pathway.

METHODSHUVECs were incubated with 0, 2.5, 5, 10, and 20 μ mol/L CPT for detecting cell viability with dimethyl thiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay. Then, HUVECs were incubated with 0, 2.5, 5, and 10 μ mol/L CPT for detecting endothelial cell migration, invasion, and tubular-like structure formation with wound healing, transwell invasion and matrigel tube formation assays, respectively. To gain insight into CPT-mediated signaling, the effects of CPT on T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcription factors were detected by the Dual-luciferase reporter assay. Next, the nuclear expression of β-catenin was evaluated using Western blot and immunochemistry. Finally, vascular endothelial growth factor (VEGF) and cyclin D1, downstream proteins of the Wnt pathway were examined with Western blot.

RESULTSCPT dose-dependently suppressed endothelial cell viability, migration, invasion, and tubular-like structure formation. In particular, CPT blocked β-catenindependent transcription in HUVECs in a dose-dependent manner. In Western bolt, 10 μ mol/L CPT decreased expression of β-catenin in nucleus of HUVECs (P<0.01). In immunohistochemistry, β-catenin was more potent in response to LiCl (an activator of the pathway) treatment. However, the signals were weaker in the nucleus of the CPT (10 μ mol/L) group, compared to the positive control. Also, VEGF and cyclin D1 were both eliminated by CPT in 5 and 10 μ mol/L doses (P<0.05).

CONCLUSIONOur study supported the role of CPT as an angiogenic inhibitor, which may impact on the Wnt/β-catenin signaling pathway.

Blotting, Western ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cyclin D1 ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Immunohistochemistry ; Luciferases ; metabolism ; Neovascularization, Physiologic ; drug effects ; Phenanthrenes ; chemistry ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism