Periodontal ligament(PDL) cells have been known as playing an important roles in periodontal regeneration and gingival fibroblasts are also important to periodontal regeneration by forming connective tissue attachment. There were rare studies about the gene expression patterns of PDL cells and gingival fibroblasts, therefore in this study, we tried cDNA microarray-based gene expression monitoring to explain the functional differences of PDL cells and gingival fibroblasts in vivo and to confirm the characteristics of PDL cells. Total RNA were extracted from PDL cells and gingival fibroblasts of same person and same passages, and mRNA were isolated from the total RNA using Oligotex mRNA midi kit(Qiagen) and then fluorescent cDNA probe were prepared. And microarray hybridization were performed. The gene expression patterns of PDL cells and gingival fibroblasts were quite different. About 400 genes were expressed more highly in the PDL cells than gingival fibroblasts and about 300 genes were more highly expressed in the gingival fibroblasts than PDL cells. Compared growth factor- and growth factor receptor-related gene expression patterns of PDL cells with gingival fibroblasts, IGF-2, IGF-2 associated protein, nerve growth factor, placental bone morphogenic protein, neuron-specific growth- associated protein, FGF receptor, EGF receptor-related gene and PDGF receptor were more highly expressed in the PDL cells than gingival fibroblasts. The results of collagen gene expression patterns showed that collagen type I, type III, type VI and type VII were more highly expressed in the PDL cells than gingival fibroblasts, and in the gingival fibroblasts collagen type V, XII were more highly expressed than PDL cells. The results of osteoblast-related gene expression patterns showed that osteoblast specific cysteine-rich protein were more highly expressed in the PDL cells than gingival fibroblasts. The results of cytoskeletal proteins gene expression patterns showed that alpha-smooth muscle actin, actin binding protein, smooth muscle myosin heavy chain homolog and myosin light chain were more highly expressed in the PDL cells than gingival fibrobalsts, and beta-actin, actin-capping protein(beta subunit), actin- related protein Arp3(ARP) and myosin class I(myh-1c) were more highly expressed in the gingival fibroblasts than PDL cells. Osteoprotegerin/osteoclastogenesis inhibitory factor(OPG/OCIF) was more highly expressed in the PDL cells than gingival fibroblasts. According to the results of this study, PDL cells and gingival fibroblasts were quite different gene expression patterns though they are the fibroblast which have similar shape. Therefore PDL cells & gingival fibroblasts are heterogeneous populations which represent distinct characteristics. If more studies about genes that were differently expressed in each PDL cells & gingival fibroblasts would be performed in the future, it would be expected that the characteristics of PDL cells would be more clear.
Actins ; Carrier Proteins ; Collagen ; Collagen Type I ; Collagen Type V ; Connective Tissue ; Cytoskeletal Proteins ; DNA, Complementary* ; Epidermal Growth Factor ; Fibroblasts* ; Gene Expression Profiling ; Gene Expression* ; Humans ; Insulin-Like Growth Factor II ; Muscle, Smooth ; Myosin Heavy Chains ; Myosin Light Chains ; Myosins ; Nerve Growth Factor ; Oligonucleotide Array Sequence Analysis* ; Osteoblasts ; Periodontal Ligament* ; Receptors, Fibroblast Growth Factor ; Receptors, Platelet-Derived Growth Factor ; Regeneration ; RNA ; RNA, Messenger

BACKGROUND: Single or multiple episodes of brief period of ischemia and reperfusion(ischemic preconditioning, IP) have been shown to limit infarct size after a subsequent longer period of ischemia. A considerable number of possible mechanisms has been proposed, however, controversies still remain. Accordingly, we evaluated the effect of four cycles of 5 minutes ischemia and 5 minutes reperfusion(IP) followed by subsequent 30 minutes ischemia(ISCH) and 60 minutes reperfusion using isolated Langendorff-Perfused rabbit hearts. Methods and RESULTS: After a 50-minute recovery phase, parameters of the left ventricular function(LVF) including left ventricular developed pressure(LVDP), contractility and the heart rate were recorded, and ultrastructure was examined. Myosin ATPase activity was determined by measurement of inorganic phosphorus and isozymes of the myosin heavy chain were examined by polyacrylamide gel electrophoresis containing pyrophosphate buffer. The ISCH hearts showed severe to irreversible change of the cardiac myocytes homogenously in contrast to the IP hearts in which changes were not homogenous and irreversible injury was only focal. However, parameters of the LVF were not significantly different between the IP and the ISHC hearts during reperfusion. Myosin ATPase activities were also not significantly different(0.67+/-0.123 micromol/mg protein/h in the IP hearts, 0.56+/-0.172 micromol/mg protein/h in the ISCH hearts, and 0.76+/-0.239 micromol/mg protein/h in the control hearts). Band patterns of the myofibrillar proteins, separated by sodium ddodecyl sulfate-polyacrylamide gel electrophoresis, revealed no differences between the IP, ISCH and the control hearts. Myosin heavy chains in the IP and the ISCH hearts were separated into 3 isozymes, V1,V2and V3in pyrophosphate gel electrophoresis in contrast that the control hearts revealed two isozymes, V1and V2. However, there were no differences in the protein composition and electrophoretic motility between the IP and the ISCH hearts. CONCLUSION: These results indicate that IP could not attenuate the changes in LVF, myosin ATPase activity and myosin isozymes on reperfusion, however, it could attenuate the ultrastructural changes of the cardiac myocytes.
Adenosine Triphosphatases* ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Heart Rate ; Heart* ; Ischemia ; Ischemic Preconditioning* ; Isoenzymes ; Myocytes, Cardiac ; Myosin Heavy Chains ; Myosins* ; Phosphorus ; Reperfusion ; Sodium ; Ventricular Function, Left*

OBJECTIVETo identify the casual mutation of a Chinese pedigree with hypertrophic cardiomyopathy (HCM), and to analyze the genotype-phenotype relationship.

METHODSThe coding exons of 26 reported disease genes were sequenced by targeted resequencing in the proband and the identified mutation were detected with bi-directional Sanger sequencing in all family members and 307 healthy controls. The genotype-phenotype correlation was analyzed in the family.

RESULTSA missense mutation (c.2191C > T, p. Pro731Ser) in the 20th exon of MYH7 gene was identified. This mutation was absent in 307 healthy controls and predicted to be pathogenic by PolyPhen-HCM. Totally 13 family members carried this mutation, including 10 patients with HCM and 3 asymptomatic mutation carriers. The proband manifested severe congestive heart failure and 8 patients expressed various clinical manifestations of heart failure, including dyspnea, palpitations, chest pain, amaurosis or syncope. Five patients were diagnosed as HCM at the age of 16 or younger. One family member suffered sudden cardiac death.

CONCLUSIONSThe Pro731Ser of MYH7 gene mutation is a causal and malignant mutation linked with familiar HCM.

Adolescent ; Asian Continental Ancestry Group ; Base Sequence ; Cardiomyopathy, Hypertrophic ; ethnology ; genetics ; Death, Sudden, Cardiac ; Exons ; Humans ; Mutation, Missense ; Myosin Heavy Chains ; genetics ; Pedigree ; Phenotype ; Research Design ; Ventricular Myosins

BACKGROUNDHypertrophic cardiomyopathy (HCM) is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain (beta-MHC) are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype.

METHODSThe exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed.

RESULTSThe mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13,619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated.

CONCLUSIONThe Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

Adolescent ; Adult ; Cardiomyopathy, Hypertrophic, Familial ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation, Missense ; Myosin Heavy Chains ; genetics ; Ventricular Myosins ; genetics

OBJECTIVE: To analyze the clinical phenotype and MYH7 gene variant in a Chinese pedigree affected with hypertrophic cardiomyopathy (HCM). METHODS: The proband was screened for variant of 96 cardiomyopathy-associated genes by exonic amplification and high-throughput sequencing. Candidate variant was verified by Sanger sequencing among 300 healthy controls as well as family members of the proband. Co-segregation analysis of genotypes and clinical phenotypes was carried out for the pedigree. Clustal X software was used to analyze the sequence conservation of the variant among various species, and its pathogenicity was predicted by using bioinformatics software. RESULTS: 6 out of 12 members from this pedigree were found to harbor heterozygous c.4124A>G (p.Tyr1375Cys) variant of the MYH7 gene, among whom five were diagnosed with HCM. The remaining one had failed to meet the diagnostic criteria for HCM, but had abnormal ECG. The same variant was not found in the 300 healthy controls. Amino acid sequence analysis showed that the variant is located in a highly conserved region, and bioinformatics analysis predicted that this variant may affect protein function and has a deleterious effect. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, the variant was predicted to be likely pathogenic (PM2+ PP1_Moderate+PP3+PP5). CONCLUSION The c.4124A>G (p.Tyr1375Cys) variant of the MYH7 gene probably underlay the pathogenesis in this pedigree. Above finding has important value for the early diagnosis of patients with HCM.
Cardiac Myosins/genetics* ; Cardiomyopathy, Hypertrophic/genetics* ; Genotype ; Humans ; Mutation ; Myosin Heavy Chains/genetics* ; Pedigree ; Phenotype

BCL-2 interacting cell death suppressor (BIS), which is ubiquitously expressed, has important roles in various cellular processes, such as apoptosis, the cellular stress response, migration and invasion and protein quality control. In particular, BIS is highly expressed in skeletal and cardiac muscles, and BIS gene mutations result in human myopathy. In this study, we show that mRNA and protein levels of BIS were markedly increased during skeletal myogenesis in C2C12 cells and mouse satellite cells. BIS knockdown did not prevent the early stage of skeletal myogenesis, but did induce muscle atrophy and a decrease in the diameter of myotubes. BIS knockdown significantly suppressed the expression level of myosin heavy chain (MyHC) without changing the expression levels of myogenic marker proteins, such as Mgn, Cav-3 and MG53. In addition, BIS endogenously interacted with MyHC, and BIS knockdown induced MyHC ubiquitination and degradation. From these data, we conclude that molecular association of MyHC and BIS is necessary for MyHC stabilization in skeletal muscle.
Animals ; Apoptosis ; Cell Death* ; Humans ; Mice ; Muscle Development ; Muscle Fibers, Skeletal ; Muscle, Skeletal* ; Muscular Atrophy ; Muscular Diseases ; Myocardium ; Myosin Heavy Chains* ; Myosins* ; Quality Control ; RNA, Messenger ; Ubiquitin ; Ubiquitination

BCL-2 interacting cell death suppressor (BIS), which is ubiquitously expressed, has important roles in various cellular processes, such as apoptosis, the cellular stress response, migration and invasion and protein quality control. In particular, BIS is highly expressed in skeletal and cardiac muscles, and BIS gene mutations result in human myopathy. In this study, we show that mRNA and protein levels of BIS were markedly increased during skeletal myogenesis in C2C12 cells and mouse satellite cells. BIS knockdown did not prevent the early stage of skeletal myogenesis, but did induce muscle atrophy and a decrease in the diameter of myotubes. BIS knockdown significantly suppressed the expression level of myosin heavy chain (MyHC) without changing the expression levels of myogenic marker proteins, such as Mgn, Cav-3 and MG53. In addition, BIS endogenously interacted with MyHC, and BIS knockdown induced MyHC ubiquitination and degradation. From these data, we conclude that molecular association of MyHC and BIS is necessary for MyHC stabilization in skeletal muscle.
Animals ; Apoptosis ; Cell Death* ; Humans ; Mice ; Muscle Development ; Muscle Fibers, Skeletal ; Muscle, Skeletal* ; Muscular Atrophy ; Muscular Diseases ; Myocardium ; Myosin Heavy Chains* ; Myosins* ; Quality Control ; RNA, Messenger ; Ubiquitin ; Ubiquitination

BACKGROUND: The most important feature distinguishing intraductal papilloma (IDP) from papillary carcinoma is the presence of uniform myoepithelial cells (MECs) within the lesion. METHODS: Immunohistochemistry (IHC) for calponin, smooth muscle myosin heavy chain (SMMHC), cytokeratin 34E12, and p53 were performed on 37 IDP, 4 intraductal papillary carcinomas (IDPCA), 5 microinvasive papillary carcinomas, and 5 invasive papillary carcinomas (IPCA), respectively. RESULTS: The mean age of the patients was 43 (43.3+/-11.6) years. Cytokeratin 34E12 was expressed in epithelial cells (84%) as well as in MECs (23%) of IDP. The expression of SMMHC was significantly reduced in the intraductal and invasive papillary carcinoma (p=0.001). The expression of calponin was also significantly reduced (P<0.001) as IDP 95%, IDPCA 76%, microinvasive papillary carcinoma 39%, and IPCA 8%, respectively. p53 over-expression was noted in 3 (one IDP and two IPCA) of 51 cases. CONCLUSIONS: Because MECs were significantly reduced with malignant progression, calponin and SMMHC were very useful markers for differentiating between benign and malignancy in the papillary neoplasm. Calponin was more sensitive than SMMHC and was an excellent ancillary test for assessing MECs and for detecting microinvasion.
Breast* ; Carcinoma, Papillary ; Epithelial Cells ; Humans ; Immunohistochemistry ; Keratins* ; Muscle, Smooth* ; Myosin Heavy Chains* ; Myosins* ; Papilloma, Intraductal ; World Health Organization

Exercise training can improve strength and lead to adaptations in the skeletal muscle and nervous systems. Skeletal muscles can develop into two types: fast and slow, depending on the expression pattern of myosin heavy chain (MHC) isoforms. Previous studies reported that exercise altered the distribution of muscle fiber types. It is not currently known what changes in the expression of caveolins and types of muscle fiber occur in response to the intensity of exercise. This study determined the changes in expression of caveolins and MHC type after forced exercise in muscular and non-muscular tissues in rats. A control (Con) group to which forced exercise was not applied and an exercise (Ex) group to which forced exercise was applied. Forced exercise, using a treadmill, was introduced at a speed of 25 m/min for 30 min, 3 times/day (07:00, 15:00, 23:00). Homogenized tissues were applied to extract of total RNA for further gene analysis. The expression of caveolin-3 and MHC2a in the gastrocnemius muscle of female rats significantly increased in the Ex group compared with the Con group (P<0.05). Furthermore, in the gastrocnemius muscle of male rats, the expression of MHC2x was significantly different between the two groups (P<0.05). There was an increased expression in caveolin-3 and a slightly decreased expression in TGFbeta-1 in muscular tissues implicating caveolin-3 influences the expression of MHC isoforms and TGFbeta-1 expression. Eventually, it implicates that caveolin-3 has positive regulatory function in muscle atrophy induced by neural dysfunction with spinal cord injury or stroke.
Animals ; Caveolin 3 ; Caveolins ; Female ; Humans ; Male ; Muscle, Skeletal ; Muscles ; Muscular Atrophy ; Myosin Heavy Chains ; Myosins ; Nervous System ; Protein Isoforms ; Rats ; RNA ; Spinal Cord Injuries ; Stroke

This paper was aimed to investigate the inhibitory effect of aconitine(AC) on angiotensin Ⅱ(Ang Ⅱ)-induced H9 c2 cell hypertrophy and explore its mechanism of action. The model of hypertrophy was induced by Ang Ⅱ(1×10-6 mol·L-1),and cardiomyocytes were incubated with different concentrations of AC. Western blot was used to quantify the protein expression levels of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),β-myosin heavy chain(β-MHC),and α-smooth muscle actin(α-SMA). Real-time quantitative PCR(qRT-PCR) was used to quantify the mRNA expression levels of cardiac hypertrophic markers ANP,BNP and β-MHC. In addition,the fluorescence intensity of the F-actin marker,an important component of myofibrils,was detected by using laser confocal microscope. AC could significantly reverse the increase of total protein content in H9 c2 cells induced by Ang Ⅱ; qRT-PCR results showed that AC could significantly inhibit the ANP,BNP and β-MHC mRNA up-regulation induced by AngⅡ. Western blot results showed that AC could significantly inhibit the ANP,BNP and β-MHC protein up-regulation induced by AngⅡ. In addition,F-actin expression induced by Ang Ⅱ could be inhibited by AC,and multiple indicators of cardiomyocyte hypertrophy induced by Ang Ⅱ could be down-regulated,indicating that AC may inhibit cardiac hypertrophy by inhibiting the expression of hypertrophic factors,providing new clues for exploring the cardiovascular protection of AC.
Aconitine ; pharmacology ; Actins ; metabolism ; Angiotensin II ; Atrial Natriuretic Factor ; metabolism ; Cardiac Myosins ; metabolism ; Cardiomegaly ; Cells, Cultured ; Humans ; Hypertrophy ; Myocytes, Cardiac ; drug effects ; Myosin Heavy Chains ; metabolism ; Natriuretic Peptide, Brain ; metabolism