OBJECTIVE To establish an in vitro screening system for activin receptor-like kinase 4,5 and 7 (ALK4,ALK5 and ALK7) inhibitors.METHODS The insect expression systems for kinase domain of ALK4,5,7 and Smad2/3 proteins were established using the Bac to Bac baculovirus expression system.The desired proteins were expressed in Sf9 insect cells and purified by GST affinity.The screening system was composed of the kinase,Smad3 protein,ATP as well as the compound.The impact of the compound on the activities of ALK kinase domains was examined by measuring the amount of remnant ATP in the system as ALKs catalyzed the phosphorylation of Smad3 protein and consumed ATP during the process.The screening conditions were optimized,and validation of the screening system was conducted using known ALKs inhibitors.RESULTS All the reconstructed Bacmids were identified to be correct by PCR and restriction enzyme digestion.All the proteins were expressed in Sf9 insect cells after transfection,and purified proteins were achieved by GST affinity purification.For the screening system,the optimized kinase concentration and Smad3 concentration were 10 mg· L-1 and the optimized ATP concentration was 10 nmol·L-1.The Z'factor for ALK4,ALK5,and ALK7 kinase inhibitors screening system was 0.71,0.51 and 0.74,respectively.The well-known ALK inhibitor SB431542 inhibited the catalytic activities of ALK4,ALK5,and ALK7 with IC50 values of 22,188 and 91 nmol· L-1,respectively.CONCLUSION The in vitro screening system for ALK4,ALK5 and ALK7 inhibitors is successfully established.

Objective To study the mechanism by which a high-intensity pulsed electromagnetic field (HIPEMF) (0.1 Hz,4 T,8 pulses) facilitates the proliferation of neural stem cells by detecting the expression of β-catenin genes and protein.Methods Neural stem cells (NSCs) were isolated from the sub-ventricular zone (SVZ) of neonatal rats and cultured in supplemented,serum-free medium for two weeks.The NSCs were then divided into an experimental group exposed to a HIPEMF for 8 pulses and a control group given sham stimulation.The gene and prorein expression of β-catenin in the NSCs were assayed by RT-PCR and Western blotting on the 1st,3rd,5th and 7th day after the stimulation.Results The NSCs' cloned spheres were round and translucent,and showed red fluorescence after staining with anti-nestin (cy3).The RT-PCR results showed β-catenin genes were highly expressed in the exposed group (significantly more than in the controls).The Western blotting showed that expression of β-catenin protein was also higher in the experimental group,especially at the 7th day after stimulation,a difference which was also statistically significant Conclusion HIPEMF at 0.1 Hz,4 T,in 8 pulse trains can promote NSC proliferation,perhaps through the Wnt/β-catenin signaling pathways.

Objective To explore the therapeutic effect of irbesartan treatment on left ventricular remodeing after acute myocardial infarction by 2D Doppler technique.Methods 85 patients with AMI were selected into the study.42 patients were randomized to conventional treatment group,43 patients to irbesartan treatment group.At first week and 26 week after AMI,the left ventricular myocardium weight(LVMW) and diastolic volume(LVDV) were measured by echocardiogram,and the parameters of left ventricular systolic and diastolic function were measured by echocardiogram.Results (1)At 26 week,LVDV and LVMW in irbesartan treatment groups were significantly decreased as compared with conventional treatment group,left ventricular eiection fraction(LVEF) in irbesartan group was increased.(2)PE(peak E-wave velocity) and PFR(peak filling rate) in irbesartan group were increased as compared with conventional group.Conclusion Irbesaratan might significantly reduce myocardial dilatation of left ventricle,decrease LVMW after AMI and improve left ventricle function.

The purpose of this study was to determine the total Bolton index (TBI) and anterior Bolton index (ABI), evaluate the clinical significance of tooth size discrepancies and the influence of gender variation of mesiodistal tooth width on Bolton Index and estimate which tooth has the greatest influence on Bolton Index. A total of 110 pairs of pretreatment dental study casts (41 male and 69 female) were selected from patients treated in Department of Orthodontics of Union Hospital in Wuhan, China. A sliding dental Vernier caliper was used for the measurement of the mesiodistal tooth width. Descriptive statistical mean values, standard deviation, standard error of the mean values, coefficient of variance and the t-test were used for the statistical analysis of the data. The study found that TBI, ABI and the total sum of teeth width had no significant difference between male and female. Male group indicated that Bolton Index (BI) was mostly influenced by the width of lower right second premolar, whereas female group showed that BI was mostly influenced by the width of both the right and left maxillary lateral incisor.

Objective To investigate the roles of somatostatin(SS)positive intemeurons in the development and compensation of temporal lobe epilepsy.Methods Piloearpine-induced epilepsy rat model was established.Immunohistochemistry method was used to detect number changes and axonal sprouting of SS positive intemeurons in different domains of the hippocampus at difierent time points.Degeneration of SS positive interneurons and their neurophils were detected by the double immunofluorescence staining with SS and Fluoro-Jade B(FJB)at 7 and 60 days after status epilepticus (SE).Results In the exoerimental rat group,the number of SS positive neurons decreased in each hippocampal domain,and it reached the lowest at 7 days post-SE(There were 11.1±3.3 in hilus,2.8±0.9 in CA1region and 1.8±0.7 in CA1region,t=13.519,9.644 and 8.808,all P<0.01).In chronic phase,the number of SS neurons gradually recovered,and exceeded the control group in CA1 area at 60 days post-SE(12.8±1.5 vs 8.8±1.3,t=-4.506,P<0.01),however,the number of SS neurons in the hilus(25.5±4.6)and CA1 area(4.8±0.8)remained significantly less than normal levels(t value were 4.691 and 3.953.both P<0.01).Increased SS positive fibers were found in the lacunosum-molecular (1m)layer and outer molecular layer of dentate gyrus after 30 days post-SE,and numerous SS positive fibers were seen threnghout the layers of area CA1 at 60 days post-SE.Double immunofluuorescence revealed that a few SS positive interneurons and fibers were also labeled by FJB in area CA1 at 7 days post-SE and in CA domain/hilus at 60 days post-SE.Conclusions SS intemeurons loss plays an important role in the development of temporal lobe epilepsy.The loss is partially caIlsed by the degeneration and death of neurons;SS positive neurophils increase within area CA1 in chronic phase may play a significant role in the generation and compensation of temporal lobe epilepsy.

Objective To investigate the possible association between two single nucleotide polymorphisms (SNPs) loci of apolipoprotein E (ApoE) gene and epileptic drug resistance in a central Chinese Han population.Methods A case control study was performed in 364 epileptic patients.According to the criteria of drug resistant epilepsy proposed by International League Against Epilepsy in 2010,143 patients were classified into drug resistant group and 221 patients into drug responsive group.The peripheral venous blood of each patient was collected for DNA extraction after clinical evaluation.The candidate ApoE SNPs loci,including rs7412 and rs769450,were genotyped by BeadChip Scanning and GoldenGate Assay following the Illumina protocols.The differences in allelic and genotypic frequency were compared between groups.Linkage disequilibrium was calculated through SHEsis platform.Results There was no significant difference for genotype or allele of rs7412 between groups.The GG genotype (OR =2.038,95％ CI 1.196-3.475,P=0.009) and G allele (OR =1.618,95％CI 1.193-2.193,P=0.002) of rs7412 were significantly more abundant in the drug resistant group.As for idiopathic epileptic patients,the GG genotype (OR =2.110,95％ CI 1.189-3.744,P =0.011) and G allele (OR =1.641,95％ CI 1.187-2.270,P =0.003) of rs7412 were still significantly more abundant in the drug resistant group.There was no linkage disequilibrium between the two loci with D' value of 0.072.Conclusion The GG genotype and G allele of ApoE rs769450 may be associated with epileptic drug resistance in a central Chinese Han population.

OBJECTIVE To evaluate the anti-tumor activities of WX-127-07,a new microtubule-tar-geting agent invitroand probe its molecular mechanism. METHODS The well-known microtubule-targe-ting anti-tumor drugs taxol,vincristine and anti-gout drug colchicine were used as positive controls. The anti-proliferation activity was examined in five different cell lines after treatment with WX-127-07(0.3 -300 nmol·L-1 )for 72 h by SRB assay. The cell cycle arrest profile was assayed by flow cytometry. The multiparameters of cytotoxicity,cell morphology,apoptosis and different signaling pathways related to tumorigenesis and inflammation were analyzed using the high content analysis platform. Tubulin tryptic digestion and competition inhibition assay for colchicine or vinblastine site were used to confirm the bind-ing site in microtubules at a molecular level. RESULTS All the tested compounds obviously inhibited the growth of A549,HepG2,HeLa,HLF and HUVEC cells. The lC50 values of WX-127-07 were 4.47±0.05, 5.18±0.08,4.90±0.19,4.10±0.16 and(5.04±0.08)nmol·L-1 respectively,lower than those of colchicine〔the lC50 values were 21. 17 ± 1. 22,14. 19 ± 0. 53,43. 80 ± 1. 64,145. 89 ± 10. 97 and( 27. 67 ± 1.79)nmol·L-1 ,respectively〕and those of vincristine〔the lC50 values were 16.51±0.36,16.76±0.33, 27.80±2.75,43.80±1.48 and(9.15±0.78)nmol·L-1 ,respectively〕,but were similar to or lower than those of taxol〔the lC50 values were 10. 68 ± 0. 61,12. 86 ± 0. 25,4. 81 ± 0. 61,102. 07 ± 15. 17 and( 3. 04 ± 0.12)nmol·L-1 ,respectively〕. High content multi-parameter analysis revealed that WX-127-07 induced a concentration-dependent microtubular depolymerization(P=0.0075)with the same pattern as colchicine and vincristine,but at a lower concentration. Both WX-127-07 and positive drugs could induce cell cycle arrest in A549 cells,increase nuclear membrane permeability and early signs of apoptosis in HepG2 cells,but neither cancer related pathways nor inflammation related pathways were affected. Microtubular competition inhibition assay showed that WX-127-07 inhibited the binding of colchicine with tubulin(P =0.0259). Tryptic digestion of tubulin-WX-127-07 premixture showed a similar electrophoretic band to that of tubulin-colchicine premixture. CONCLUSION WX-127-07 is a novel microtubule-depolymerizing agent with anti-proliferation activity and acting on the colchicine binding site.

Objective To screen for selective transforming growth factor β(TGF-β)inhibitors from the compound library, and analyze their structure-activity relationship. Methods The inhibiting activities of 170 compounds to TGF-βpathway were evaluat-ed by the SMAD3 luciferase reporter system;the positive hits were examined for their selectivity towards activin receptor like kinase (ALK)4、ALK5 or ALK7 by a molecule based screening system composed of SMAD3,ATP and the purified kinase domain for ALK4, ALK5 or ALK7;the EGFP-SMAD2 fusion protein redistribution assay was used to confirm the inhibiting effects of positive hits. The structure-activity relationship was analyzed by comparing the docking module of SB431542 with ALK5 kinase domain. Results Fif-teen compounds were found capable of inhibiting luciferase expression downstream of SMAD3 with≥25%inhibitory rate;several of them showed different selectivity towards ALK4,ALK5 and ALK7. Compound 63 selectively inhibited the activity of ALK4 and ALK7 with IC500.234 and 0.370μmol/L,respectively,while compound 64 showed inhibiting activity towards all three kinases with the IC50 values 10,6 and 85 nmol/L for ALK4、ALK5 and ALK7,respectively. In addition,compounds 63 and 64 further inhibited the TGF-β1 induced EGFP-SMAD2 nuclear translocation,with the IC50 values of 0.45 and 6.30μmol/L,respectively. The MTT anti-proliferative assay indicated that compounds 63 and 64 exerted these activities at non-toxic concentrations. The analysis of structure-activity rela-tionship indicated that the compounds sharing a core structure,the 1,2,4-triarylimizazole or 1,3,5-triarylpyrazoline,with the 3,4 methyoenedioxyphenyl,6-methylpyridine and 4-aminocarboxyl substitution groups tended to exhibit better activities. Conclusion The two potent TGF-βpathway inhibitors,63 and 64 are identified through this screening project,of which,63 selectively inhibited the ALK4 and ALK7 activity,while 64 showed inhibiting activity towards all three tested types of ALKs.

Objective　To study the clinica features, diagnosis and surgical treatment of simple and complex macromastia. Methods　The clinical and pathological data of 21 patients with macromastia in our hospital from 1990 to 2000 were analysed retrospectively. Simple macromastia in 16 patients with 29 breasts, complex macromastia in 5 patients with 8 breasts. Results　The average age of patients was 23 years old. All the 21 patients were treated surgically. Among the 5 patients with complex macromastia, subcutaneous mastectomy and reconstruction were performed on 2 patients with breast fibroadenomatosis; of the 2 patients with giant fibroadenoma, one underwent tumor excision, the others were subjected to tumor excision with reduction mammaplasty; the last one with breast fibrous disease, selective partial breast excision and reduction mammaplasty was performed. All the patients had satisfactory mammary appearance and hadn't recurrrence of the breast disease in follow up period. Conclusions　(1) Macromastia often happens in the adolescent and young women. (2) It is necessary to do biopsy during operation for distinguishing the simple from the complex macromastia. (3) The treatment for simple macromatia is reduction mammaplasty; for complex macromatia is dissection of the disease area completely and redution mammaplasty simultaneously. For macromastia with fibroadenomatosis, subcutaneous mastectomy with reconstruction can prevent tumor recurrence and get better mammary appearance.

Objective To investigate the effects of artesunate (Art) on cell apoptosis, tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) expression induced by high glucose in rat renal tubular epithelial cells (NRK-52E). Methods NRK-52E cells were cultured and divided into normal control group, high glucose group, high glucose with different concen?trations of Art (10 mg/L, 20 mg/L and 30 mg/L) groups, and high glucose with Ena (5 mg/L) group. MTT assay was used to de?tect the cell proliferation. The apoptotic rate was evaluated by flow cytometry with AnnexinV-FITC/PI double stains. The pro?tein levels of TNF-αand IL-8 in the cell culture supernatant were determined using ELISA. Results High glucose inhibit?ed NRK-52E proliferation, induced its apoptosis, and the expressions of TNF-αand IL-8 in the supernatant. Application of Art obviously abolished the effects of high glucose, and the effects of Art were showed in dose-dependent manner. Conclu?sion Art can suppress high glucose-stimulated cell apoptosis, enhance TNF-αand IL-8 expression in NRK-52E cells. The anti-inflammatory action and immune regulation of Art could be a novel approach of treating diabetic nephropathy.