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Objective: To explore the protective effect of electrical stimulation of phrenic nerve on diaphragmatic function during mechanical ventilation. Methods: Forty healthy adult SD rats were randomly divided into 5 groups: blank control group (BC), spontaneous breathing group (SB), electrical stimulation group (ES), mechanical ventilation group (MV), and electrical stimulation and mechanical ventilation group (MS). The rats in each group were treated for 18 h except for the BC group. After treatment, the diaphragm muscle tissue was obtained and the diaphragm contractility including peak-to-peak value(Vpp) and maximum rate of contraction(+ dT/dt max) were measured. Expression of MyoD and myogenin were detected. Results: Except for the ES and the MS groups, there was a significant difference for peak-to-peak value (Vpp) between each 2 groups ( P <0.05). Expression levels of MyoD in treatment groups were also significantly different ( P <0.05). Expressions of MS(Q-PCR 2(-ΔΔCt) value: 11.66±2.80) and MV(Q-PCR 2(-ΔΔCt) value: 40.89±24.71) in the treatment group were significantly different ( P <0.05). The expression of myogenin in the MS and the MV groups were significantly different from those of the BC group( P <0.05), however there was no significant difference between the MS(Q-PCR 2(-ΔΔCt) value: 2.58±2.75) and the MV group(Q-PCR 2(-ΔΔCt) value: 1.63±0.71). Conclusions: Electrical stimulation of the phrenic nerve can change the expression level of MyoD and myogenin to offset mechanical ventilation induced diaphragmatic function damage, and therefore plays a protective effect on the diaphragm.

Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.

As one of the members of MADS family, MEF2 group is important in regulating development. Analytical tools of NCBI, ExPASy, CBS, CDD, and SABLE were adopted to analyze the properties of human MEF2 proteins, and evolutionary tree was built according to the result of correlative sequence alignments. The results showed that there are various forms of MEF2 in human body, and there are some differences in the physicochemical characteristics. Relatively more phosphorylation sites are found and the main glycosylation sites are N-glycosylation sites. All MEF2 proteins of human contain MADS domain, and most contain MEF2 domain and HJURP&#95;C domain. Their secondary structures contain three dominant states: helix, sheet and coil, their tertiary structures are similar. The phylogenetic tree result shows that MEF2B may be original because of its difference of sequences and evolutional relation.

Objective: To investigate the association of the 482G/A polymorphism of the PGC-1alpha gene with type 2 diabetes by family-based study in the Han population in South China, and to analyze the quantitative and qualitative binding force changes between the PGC-1alpha domain mutant and MEF2C, as well as to evaluate the possibility of PGC-1alpha -MEF2C-GLUT4 pathway in the pathogenesis of type 2 diabetes., Methods: Blood samples were collected from 350 patients with type 2 diabetes and their first-degree relatives. Genomic DNA was extracted and polymorphic PGC-1alpha genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism and direct DNA sequencing. The results were analyzed by family-based transmission disequilibrium test (TDT) and haplotype relative risk (HRR). The protein-protein interaction between PGC-1alpha and MEF2C was detected by means of the site-directed mutagenesis kit and bacteriomatch two-hybrid system kit., Results: In the family-based study, HRR analyses demonstrated that the 482A allele was more often transmitted to patients than predicted by chance (chi (2)= 7.2170, P= 0.0072, HRR= 1.4496). TDT-extended test(ETDT) analyses also revealed that PGC-1alpha 482A allele was significantly deviated from 0.5 from heterozygous parents to patients than expected (219 trios, P= 0.0310; 350 trios, P= 0.0292). BacterioMatch Two-Hybrid System showed that 482A variation could lead to decreased binding force between PGC-1alpha and MEF2C (62.1+/- 8.97, P< 0.05)., Conclusion: The 482A polymorphism increases the risk of developing type 2 diabetic mellitus in the South China Han population, which might be mediated by the PGC-1alpha -MEF2C-GLUT4 pathway.

The population genetic information about genetic distribution, variation and heterozygosity of Myf6 gene in five purelines and three crossbreds of chicken was analyzed using PCR-SSCP. And the genetic effects of Myf6 gene on fiber density and partial carcass traits were investigated. Only one mutation G/A at base position 47 of the whole CDS was found among individuals in each line. The distribution of Myf6 genotype strayed off the Hardy-Weinberg equilibrium and the heterozygosity was low in two purelines but not in three crossbreds. The wild-type allele A had significant positive effects on live-weight, carcass-weight, breast-muscle-weight and leg-muscle-weight (P<0.05), but had negative effects on most of meat quality traits. The A allele had additive effects of increasing 45.54 g of the live-weight, 41.03 g of the carcass-weight, 6 g of the breast-muscle-weight, 9.775 g of the leg-muscle-weight. The A allele had dominant effects of increasing 1.025 mm of the diameter of muscle fiber, -29.99 mm2 of the density of muscle fiber.

Single nucleotide polymorphisms (SNPs) of hircine myf-5, myf-6 genes and 5'flanking region of the myoD gene (myoD 5') were analyzed by PCR-SSCP in Boer goat and Xuhuai white goat. Three genotypes were found at the myf-5 locus of Xuhuai white goat, denoted AA, AB and BB, while all of Boer goats were of AA genotype. Only AA and AB genotypes were found at myf-6 and myoD 5' loci. The effects of genotypes of myf-5, myf-6 and myoD 5'flanking region loci on body measurement traits were estimated. The results showed that genotypes at the myf-5 locus were significantly associated with hucklebone width and hucklebone width index in Xuhuai white goat (P<0.05). Genotypes at the myf-6 locus were significantly associated with withers heights and hucklebone width index in Boer goat (P<0.05), but no significant associations were found for any traits in Xuhuai white goat. The myoD 5' locus was not significantly associated with any traits in both types of goat.

Objective: To analyze distribution characteristics of PGC-1alpha gene coding single nucleotide polymorphisms (cSNPs), and to investigate the association between cSNPs and type 2 diabetes mellitus, and to study biological information about PGC-1alpha domain muscle enhancer factor 2C (MEF2C) in Chinese Han population., Methods: These cSNPs were identified by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA direct sequencing technology in a total of 263 type 2 diabetic patients and 282 normal glucose tolerant controls. The possible association was analyzed between type 2 diabetes mellitus and the specific cSNPs and their haplotypes by case-control method. The tertiary structure of PGC-1alpha domain MEF2C was predicated and analyzed for possible biological information by a series of bioinformatics soft wares., Results: Four variants were found in whole extron-wide of PGC-1alpha gene in Chinese Han diabetic population. They were 394G/A, 482G/A, 528A/G and 612C/T. The 482G/A polymorphism was remarkably associated with type 2 diabetes (chi2 = 14.2025, P= 0.0002). Haplotypes analysis shown that distribution frequency of haplotypes had a statistical difference between type 2 diabetes mellitus and normal glucose tolerance control groups (chi2 = 59.9, P< 0.01) and haplotype 394A-482A-528A had a linkage disequilibrium with type 2 diabetes (t= 2.361, P< 0.05). The tertiary simulant structure of PGC-1alpha domain MEF2C was established successfully by computer. The 482G/A variant accompanied with hydrogen bonds breaking might decrease hydrophobicity and lead to an incompact space configuration which was very critical to function., Conclusion: The 482G/A variant could decrease binding force between PGC-1alpha and MEF2C and increase the risk of type 2 diabetes in Chinese Han population by PGC-1alpha -MEF2C-GLUT-4 pathway.

Objective: To explore the mutations of MEF2A gene in Chinese patients with coronary artery disease(CAD)., Methods: With polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA direct sequencing, the mutation analysis of exon 11 of MEF2A gene was performed to 156 patients with CAD and 93 normal controls., Results: By DNA sequence analyzing the samples of abnormal mobility shift of SSCP, the MEF2A gene mutations were found in three patients with CAD. One of mutations was 147130(C>A)(P431Q), and the second one was 21 bases deletion(147108-147128) which was leading to the absence of 7 amino acids (424QQQQQQQ430), and the third was 147191(G>T). Three mutations were all found in one patient, but meanwhile 21 bases deletion was found in the other two patients., Conclusion: Mutations in exon 11 of MEF2A gene exist in the patients with CAD, and the mutations may be pathological.

As a member of MRFs, Myf-6 gene plays an important role in the progress of myogenesis. The PCR-RFLP technique was applied in this study to analyze the distribution of Myf-6 gene in 12 different pig breeds and hybrid breeds. The genetic effects of Myf-6 gene on muscle fiber, carcass quality, carcass grading traits and meat quality traits were analyzed. The results indicated that: The polymorphisms of Myf-6/Ava I genotypes (intron1) were not abundant. Most of Chinese native pigs were AA homozygotes. B allele can be found only in foreign pig breeds and hybrid pigs originated from foreign pig breeds. Although no BB individuals were found in the experiment, the carcass lean percent of AB individuals (50.344 %) was significantly higher than that of AA individuals (45.875 %)(P<0.01), the loin eye area of AB individuals (27.097 cm2) was significantly larger than that of AA individuals (22.572 cm2)(P<0.01), and the carcass fat content of AB individuals (39.889 %) was significantly lower than that of AA individuals (44.503 %)(P<0.01). That means that the B allele can increase carcass lean percent and loin eye area, and decrease the carcass fat content, thus cause better carcass quality. No significant effects of the genotypes were found on the muscle fiber, FOM carcass grading traits and meat quality traits (P>0.05).

Background & Objective: There were a few reports about the role of PAX3-FKHR fusion gene in the oncogenesis of alveolar rhabdomyosarcoma (ARMS); however, its effect on muscle-producing factor (MPF) is not clear. This study was designed to observe the effects of fusion gene PAX3-FKHR transfection on expression of MRF4 and differentiation of cultured human rhabdomyosarcoma RD cells., Methods: To construct an eukaryotic expression vector of PAX3-FKHR gene; the plasmids of MRF4 cDNA and PAX3-FKHR cDNA were transfected into cultured RD cells with lipofectin methods. The changes of cells growth, morphologic and the expression of MHC and alpha-actin were observed., Results: There was no significant changes in RD cells after transfection with PAX3-FKHR gene in growth rate, growth manner and the expression of MHC and alpha-actin. In MRF4 expressing RD cells after transfected with PAX3-FKHR, although the expression of MRF4 was not changed, morphologic appearance showed a low differentiation; expression of MHC and alpha-actin significantly decreased., Conclusion: There is no obvious influence on biological character and differentiation in RD cells with transfection of PAX3-FKHR gene; PAX3-FKHR does not inhibit the expression of MRF4, but can reverse the effect of MRF4 on promoting the differentiation of RD cell.

Objective: To construct eukaryotic expression plasmid of mouse myogenic regulatory factor MyoD gene for further study on MyoD gene function in molecular regulatory mechanism in skeletal muscle repair., Methods: The plasmids PEMMBC2 beta 5 containing full cDNA length of MyoD inserted in EcoRI restriction site, were first propagated in Escherichia coli DH5a, then extracted and purified with the Wizard Plus Minipreps DNA Purification System (Promega, USA). The coding sequence of MyoD in PEMMBC2 beta 5 was confirmed by agarose gel electrophoresis and DNA sequence analysis. After plasmids PEMMBC2 beta 5 and plasmids pcDNA3-neo were prepared by digestion with EcoRI, the MyoD cDNA fragment was inserted into EcoRI site in pcDNA3-neo eukaryotic expression vector, and pcDNA3/MyoD was formed. The pcDNA3/MyoD, digested with restriction enzymes, was found to contain the MyoD cDNA sequence by agarose gel electrophoresis analysis., Results: The extracted and purified PEMMBC2 beta 5 contained the correct nucleotide sequence for the full length of MyoD cDNA fragment. The MyoD cDNA fragment had been inserted into the eukaryotic expression plasmid pcDNA3-neo, which formed the pcDNA3/MyoD., Conclusion: The pcDNA3/MyoD, a eukaryotic expression plasmid, for MyoD is constructed successfully.