OBJECTIVETo explore the effects of microtubule depolymerization (MD) on the spontaneous beating rate, action potential (AP), and oxygen consumption of cardiac myocytes in rats and its mechanism.

METHODSOne-hundred and eighty neonatal SD rats divided into 12 batches were used in the experiment, and 15 rats in each batch were sacrificed for the isolation and culture of cardiac myocytes after the heart tissues were harvested. The cardiac myocytes were respectively inoculated in one 12-well plate filled with 6 round cover slips, one 12-well plate filled with 6 square cover slips, two cell culture flasks, and two cell culture dishes. After routine culture for three days, the cardiac myocytes from all the containers were divided into normal control group (NC, routinely cultured with 3 mL DMEM/F12 solution rewarmed at 37 °C for 3 h) and group MD (routinely cultured with 3 mL DMEM/F12 solution rewarmed at 37 ° and containing 8 µmol/L colchicine for 3 h) according to the random number table, with 3 holes, 1 flask, or 1 dish in each group. The morphological changes in microtubules were observed with confocal laser scanning microscope after immunofluorescent staining. The content of polymerized or dissociative α-tubulin was determined by Western blotting. Spontaneous beating rate of the cells was observed and calculated under inverted microscope. Dissolved oxygen concentration of DMEM/F12 solution containing cardiac myocytes was determined by oxygen microelectrode system before and after the addition of colchicine. Additionally, dissolved oxygen concentration of DMEM/F12 solution and colchicine + DMEM/F12 solution was determined. The whole-cell patch-clamp technique was used to record AP, delayed rectifier K+ current (I(K)), and L-type Ca2+ current (I(Ca-L)) in cardiac myocytes; current density-voltage (I-V) curves were drawn based on the traces. Data were processed with independent or paired samples t-test.

RESULTS(1) In group NC, microtubules of cardiac myocytes were around the nucleus in radial distribution with intact and clear linear tubiform structure. The microtubules in group MD were observed in dispersive distribution with damaged structure and rough linear tubiform structure. (2) In group MD, the content of dissociative α-tubulin of cells (0.61 ± 0.03) was obviously higher than that in group NC (0.46 ± 0.03, t = -6.99, P < 0.05), while the content of polymerized α-tubulin (0.57 ± 0.04) was significantly lower than that in group NC (0.88 ± 0.04, t = 9.09, P < 0.05). (3) Spontaneous beating rate of cells was (59 ± 8) times per min in group MD, which was distinctly higher than that in group NC [(41 ± 7) times per min, t = 5.62, P < 0.01]. (4) Dissolved oxygen concentration of DMEM/F12 solution containing cardiac myocytes was (138.4 ± 2.5) µmol/L, and it was reduced to (121.7 ± 3.6) µmol/L after the addition of colchicine ( t = 26.31, P < 0.05). There was no obvious difference in dissolved oxygen concentration between DMEM/F12 solution and colchicine + DMEM/F12 solution (t = 0.72, P > 0.05). (5) Compared with that of group NC, AP morphology of cells in group MD changed significantly, with unobvious repolarization plateau phase and shorter action potential duration (APD). The APD20, APD50, and APD90 were respectively (36.2 ± 3.8), (73.7 ± 5.7), and (115.1 ± 8.0) ms in group MD, which were significantly shorter than those of group NC [(40.2 ± 2.3), (121.4 ± 7.0), and (169.4 ± 5.6) ms, with t values respectively 2.61, 15.88, and 16.75, P values below 0.05]. (6) Compared with that of group NC, the I-V curve of I(K) of cells in group MD moved up with higher current density under each test voltage (0 to 40 mV) after activation ( with t values from 2. 70 to 3. 76, P values below 0.05) . (7) There was not much alteration in current density of I(Ca-L) under each test voltage (-30 to 50 mV) between 2 groups (with t values from -1.57 to 1.66, P values above 0.05), and their I-V curves were nearly overlapped.

CONCLUSIONSAfter MD, the I(K) is enhanced without obvious change in I(Ca-L), making AP repolarization faster and APD shortened. Then the rapid spontaneous beating rate increases oxygen consumption of cardiac myocytes of rats.

Action Potentials ; Animals ; Cells, Cultured ; Energy Metabolism ; Microtubules ; metabolism ; Mitochondria, Heart ; metabolism ; Myocytes, Cardiac ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tubulin ; metabolism

OBJECTIVE To investigate the contribution of the GJB6 gene [encoding connexin 30 (C?30)] mutations in Chinese population with sporadic non-syndromic hearing impairment. METHODS PCR reactions were performed with two pair of primers for the coding sequence of GJB6 gene and for the deletion of GJB6. PCR products bidirectional sequencing was subsequently applied in 214 patients with hearing loss and 86 normal controls. RESULTS A novel heterozygous mutation-233(C→A) was found, which results in amino acid change, A78D. This mutation wasn't detected in the control subjects. The altered valine residue lies within the second conserved transmembrane domain. The large deletion△(GJB6/ D13S1830)] of GJB6 was not found in this group. CONCLUSION The large deletion of GJB6 was not found in the Chinese deafness population. A novel heterozygous mutation of GJB6 was found. These results indicated GJB6 mutations are not a major cause of hearing loss in the Chinese population.

To study the chemo-preventive effect of the supercritical extracts from Angelica sinensis (SFE-AS) on induced colorectal carcinoma in mice by using the AOM/DSS-induced male mice colorectal carcinoma model, and discuss its possible action mechanism. Male Balb/c mice were subcutaneously injected with single dose of azoxymethane (AOM, 10 mg x kg(-1) body weight). One week later, they were given 2% dextran sodium sulfate (DSS) in drinking water for 7 days to induce colorectal carcinoma. Each drug group was orally administered with supercritical extracts from Angelica sinensis at 15, 30, 60 mg x kg(-1) until the 17th week. The tumor incidence rate of the SFE-AS group, mice tumor-bearing quantity and tumor-bearing volume of the SFE-AS group were lower than that of the AOM/DSS model control group, which may be related with the significant reduction of PCNA, COX-2, iNOS in the AOM/DSS-induced mouse colorectal carcinoma model associated with inflammation by SFE-AS. According to the results of this study, SFE-AS showed an intervention effect in the incidence and development of AOM/DSS-induced mouse colorectal carcinoma associated with inflammation, and could be further used in chemo-preventive studies on human colorectal carcinoma.
Angelica sinensis ; chemistry ; Animals ; Azoxymethane ; adverse effects ; Colonic Neoplasms ; chemically induced ; genetics ; immunology ; prevention & control ; Colorectal Neoplasms ; chemically induced ; genetics ; immunology ; prevention & control ; Cyclooxygenase 2 ; genetics ; metabolism ; Dextran Sulfate ; adverse effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Proliferating Cell Nuclear Antigen ; genetics ; immunology

12E7 Antigen ; Adolescent ; Adult ; Antigens, CD ; metabolism ; Biomarkers, Tumor ; metabolism ; Brain Neoplasms ; secondary ; Cell Adhesion Molecules ; metabolism ; Child ; Child, Preschool ; Diagnosis, Differential ; Extremities ; Female ; Follow-Up Studies ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Infant ; Ki-67 Antigen ; metabolism ; Male ; Neuroectodermal Tumors, Primitive ; metabolism ; pathology ; Oncogene Proteins, Fusion ; metabolism ; Repressor Proteins ; metabolism ; Sarcoma, Ewing ; metabolism ; pathology ; Sarcoma, Synovial ; diagnosis ; metabolism ; pathology ; surgery ; Soft Tissue Neoplasms ; diagnosis ; metabolism ; pathology ; surgery ; Vimentin ; metabolism ; Young Adult

Gene Expression Profiling ; Humans ; Killer Cells, Natural ; pathology ; Lymphoma, T-Cell ; genetics ; metabolism ; pathology ; Nose Neoplasms ; genetics ; metabolism ; pathology ; Oligonucleotide Array Sequence Analysis ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the association of the polymorphism of I181L, V183L and E237G in the high affinity immunoglobulin E receptor beta-chain (FcepsilonR1beta) with the susceptibility of childhood asthma and the serum total immunoglobulin E (IgE) level.

METHODSThe coding variants of I181L, V183L and E237G and the serum total IgE level were detected using amplification refractory mutation systemdouble ended arrowpolymerase chain reaction (ARMS-PCR) and double antibody sandwich ELISA respectively in 50 asthmatic children and 40 normal controls from Sichuan Province. The association of gene mutation with the susceptibility of asthma and the serum total IgE level was analyzed.

RESULTSThere were 5 cases of I181L mutation, 2 of V183L mutation, and 7 of E237G mutation in the Asthmatic group. There was no mutation in the Normal control group. The frequency of I181L and E237G mutation in the Asthmatic group were statistically higher than in the Normal control group (P < 0.01). The serum total IgE level in the Asthmatic subgroup with I181L mutation (2.837 +/- 0.407) or E237G mutation (3.044 +/- 0.419) was significantly higher than in the Asthmatic subgroup without gene mutation (2.156 +/- 0.638) and the Normal control group (1.348 +/- 1.291) (P < 0.05 or 0.01).

CONCLUSIONSThe polymorphism of Fc epsilonR1betaI181L and E237G is a susceptible gene of childhood asthma and closely associates with the increased serum total IgE level.

Adolescent ; Asthma ; genetics ; immunology ; Child ; Child, Preschool ; Female ; Genetic Predisposition to Disease ; Humans ; Immunoglobulin E ; blood ; Infant ; Male ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Receptors, IgE ; genetics

OBJECTIVETo explore the effects of ytterbium citrate on human liver carcinoma HepG2 cell line and the potential mechanisms.

METHODSThe HepG2 cells were cultured with DMEM medium and divided into different groups in the following media, in serum-free medium as control, different concentration (0.01 - 5.00 mmol/L) [YbCit(2)](3-)+serum-free medium as treatment group, MTT assay was used to measure the viability of the cells; 2.00 mmol/L [YbCit(2)](3-)+serum-free medium was used as treatment group, and Hoechst 33258 staining was used to detect apoptosis in HepG2 cells. Differential proteomic analysis, assay of intracellular H(2)O(2) levels and mitochondrial transmembrane potential were performed to study the effects of [YbCit(2)](3-) on HepG2 cells and the potential mechanisms.

RESULTSThe data showed that 72 h treatment of [YbCit(2)](3-) at 2.00 - 5.00 mmol/L significantly inhibited cell proliferation, and the IC(50) was (2.46 ± 0.23) mmol/L. After treatment with 2.00 mmol/L [YbCit(2)](3-) for 48 h and 72 h, Hoechst 33258 staining demonstrated that [YbCit(2)](3-) induced significantly increased apoptosis in HepG2 cells. After treatment with 2.00 mmol/L [YbCit(2)](3-) for 72 h, two dimensional gel electrophoresis and MALDI-TOF mass spectrometry analysis revealed 14 differentially expressed proteins between [YbCit(2)](3-)-treated cells and the control cells. These proteins mainly included cofilin1, peroxiredoxin6, S100 calcium-binding protein A6, and proteasome 26S non-ATPase subunit 13 isoform 3 and so on. These proteins played important roles in the processes of anti-apoptosis, oxidation reduction, cell proliferation and protein degradation. The mitochondrial membrane potential were investigated, the results showed the red and green fluorescence ratio was 2.45 ± 0.28 in the control group, 1.56 ± 0.23 in 24 h group, 1.16 ± 0.18 in 48 h group, compared with the control, the differences were significant (F = 23.97, P = 0.001). The results of H(2)O(2) detection showed the fluorescence intensity was 20.00 ± 2.08 in the control group, 40.00 ± 5.50 in 24 h group, and 48.00 ± 2.03 in 48 h group, compared with the control, the differences were significant (F = 48.40, P = 0.000). The results indicated a significant reduction in mitochondrial transmembrane potential and significant increase in H2O2 generation were observed in [YbCit(2)](3-)-treated cells.

CONCLUSIONThese results suggested that [YbCit(2)](3-) could induce apoptosis of HepG2 cells through the mechanisms involving oxidative stress and mitochondrial dysfunction.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Oxidative Stress ; Proteome ; analysis ; Proteomics ; Ytterbium ; pharmacology

OBJECTIVEReceptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a calcitonin gene related peptide (CGRP) receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an adrenomedullin(ADM) receptor. As CGRP and ADM may play a beneficial role in heart failure, this study aimed at the question whether RAMPs mRNAs are changed in heart failure.

METHODSSemi-quantitative reverse transcription-PCR (RT-PCR) was used to detect and quantify the mRNAs of RAMP1 and RAMP3 in the atria of heart failing patients.

RESULTSIt was found that the expressions of RAMP1, RAMP2 and RAMP3 mRNAs increased with the worsening of heart function, but the expressions of RAMP1 and RAMP2 mRNA decreased at level IV of heart failure.

CONCLUSIONThe above results demonstrated in the atria of heart failure patients an up-regulation of CGRP receptor by an increase of RAMP1 in association with CRLR and an up-regulation of ADM receptor by an increase of RAMP2 expression in association with CRLR, thus suggesting that CGRP and ADM receptors be playing a functional role in compensating the chronic heart failure in human.

Adult ; Calcitonin Receptor-Like Protein ; Female ; Heart Atria ; metabolism ; Heart Failure ; genetics ; physiopathology ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Male ; Membrane Proteins ; genetics ; physiology ; Receptor Activity-Modifying Protein 1 ; Receptor Activity-Modifying Protein 2 ; Receptor Activity-Modifying Protein 3 ; Receptor Activity-Modifying Proteins ; Receptors, Adrenomedullin ; Receptors, Calcitonin ; genetics ; physiology ; Receptors, Calcitonin Gene-Related Peptide ; genetics ; physiology ; Receptors, Peptide ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo assess the association of smoked meat intake, SULT1A1 polymorphism as well as their combined effects with breast cancer risk.

METHODSA total of 400 newly diagnosed breast cancer cases from a cancer hospital in Sichuan province and 400 healthy controls from participants of physical examination in a hospital in Chengdu city were recruited from May 2007 to July 2009. A valid questionnaire was designed to collect their demographic characteristics and breast cancer risk factors. Daily intake of foods was collected using semi-quantitative frequency questionnaire and then the daily intake of smoked meat was calculated and transformed to energy-adjusted smoked meat intake by the residual method. Gene sequencing was used to analyze SULT1A1 Arg213His genotypes. Multivariable conditional logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (95%CIs).

RESULTSThe energy-adjusted daily intake of smoked meat (Median (P₂₅, P₇₅)) was 8.65 (3.63, 18.44) g/d in cases and 4.44 (0.19, 8.71) g/d in controls. The frequency of SULT1A1 variant allele was 14.75% (59/400) among cases and 12.75% (51/400) among controls. High energy-adjusted daily intake of smoked meat (≥ 4.44 g/d) was significantly associated with breast cancer risk among premenopausal (OR = 2.31, 95%CI: 1.46 - 3.66) and postmenopausal subjects (OR = 3.13, 95%CI: 1.89 - 5.17). High energy-adjusted daily intake of smoked meat combined with carrying SULT1A1 variant allele elevated breast cancer risk among premenopausal (OR = 3.31, 95%CI: 1.66 - 6.62) and postmenopausal subjects (OR = 3.81, 95%CI: 1.79 - 8.10).

CONCLUSIONHigh smoked meat intake contributes to high risk of breast cancer. SULT1A1 variant allele increases breast cancer risk among subjects who were exposed to high smoked meat intake.

Adult ; Arylsulfotransferase ; genetics ; Breast Neoplasms ; etiology ; genetics ; Case-Control Studies ; Cooking ; Diet ; Female ; Genotype ; Humans ; Meat ; adverse effects ; Middle Aged ; Polymorphism, Single Nucleotide ; Risk Factors

Objective To investigate the long-term efficacy of PEG-IFN alpha-2a (PEG-IFNα-2a)plus ribavirin (RBV)in treatment of chronic hepatitis C (CHC)patients with IL28B single nucleotide polymorphisms (SNPs)rs12979860 C/C type in different HCV genotypes.Methods A prospective study was conducted on 38 CHC patients from our hospital's Infection Department from March 2011 to September 2015.The patients were treated with PEG-IFNα-2a/RBV for 48 weeks.A 42-month follow-up of patients was performed after withdrawal of treatment.The main paramenters to value the efficacy were liver function,blood lipids,and sustained virological response (SVR).Results In the CHC patients with IL28B SNP rs12979860 C/C type,the rate of SVR in patients with antiviral therapy had no significant difference between groups 1b and 2a (73.33% and 95.65%,respectively, P＞0.05).After anti-HCV therapy,liver function indices such as ALT,AST,TBIL,TC,TG and HDL all significantly improved in the two groups (all P＜0.05).However,there was no difference in biochemical indices (ALT,GGT,bilirubin,blood lipids)between the two groups (all P＞0.05).Conclusion In CHC patients with IL28B SNP rs12979860 C/C type,the long-term efficacy of PEG-IFNα-2a/RBV is good.IFN-based antiviral therapy has a higher SVR rate,and liver function and lipid metabolism can be significantly improved.