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 AND OBJECTIVES: Most studies in cardiac regeneration have explored bone marrow mesenchymal stem cells (BM-MSC) with variable therapeutic effects. Amniotic fluid MSC (AF-MSC) having extended self-renewal and multi-potent properties may be superior to bone marrow MSC (BM-MSC). However, a comparison of their cardiomyogenic potency has not been studied yet.METHODS: The 5-azacytidine (5-aza) treated AF-MSC and BM-MSC were evaluated for the expression of GATA-4, Nkx2.5 and ISL-1 transcripts and proteins by quantitative RT-PCR and Western blotting, respectively as well as for the expression of cardiomyogenic differentiation markers cardiac troponin-T (cTNT), beta myosin heavy chain (βMHC) and alpha sarcomeric actinin (ASA) by immunocytochemistry.RESULTS: The AF-MSC as compared to BM-MSC had significantly higher expression of GATA-4 (183.06±29.85 vs. 9.80±0.05; p<0.01), Nkx2.5 (8.3±1.4 vs. 1.82±0.32; p<0.05), and ISL-1 (39.59±4.05 vs. 4.36±0.39; p<0.01) genes as well as GATA-4 (2.01±0.5 vs. 0.6±0.1; p<0.05), NKx2.5 (1.9±0.14 vs. 0.8±0.2; p<0.01) and ISL-1 (1.7±0.3 vs. 0.9±0.1; p<0.05) proteins. The AF-MSC also had significantly elevated expression of cTNT (5.0×10⁴±0.6×10⁴ vs. 3.5 ×10⁴±0.8×10⁴; p<0.01), β-MHC (15.7×10⁴±0.9×10⁴ vs. 8.2×10⁴±0.6×10⁴; p<0.01) and ASA (18.6×10⁴±4.9×10⁴ vs. 13.1×10⁴±3.0×10⁴; p<0.05) than BM-MSC.CONCLUSIONS: Our data suggest that AF-MSC have greater cardiomyogenic potency than BM-MSC, and thus may be a better source of MSC for therapeutic applications in cardiac regenerative medicine.
Actinin ; Amniotic Fluid ; Antigens, Differentiation ; Azacitidine ; Blotting, Western ; Bone Marrow ; Female ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; Regeneration ; Regenerative Medicine ; Therapeutic Uses ; Troponin T ; Ventricular Myosins

BACKGROUND AND OBJECTIVES: We investigated the effects of different concentrations of serum, 5-azacytidine, and culture time on the cardiomyogenic differentiation of P19 embryonal carcinoma stem cells in the course of developing an efficient protocol for generating the cardiomyogenic lineage. MATERIALS AND METHODS: P19 cells were plated at a density of 1x10(6) cells on 10-cm bacterial dishes for 96 hours in the presence of 1% dimethyl sulfoxide to form embryoid bodies. The embryoid bodies were cultured in medium with 2% or 10% fetal bovine serum for an additional 10 or 15 consecutive days in the presence of 0, 1, or 3 microM 5-azacytidine. RESULTS: Quantitative real-time polymerase chain reaction (PCR) analysis showed that the messenger ribonucleic acid (mRNA) expression of cardiac muscle-specific genes, such as GATA4, alpha-actin, alpha-myosin heavy chain, and cardiac troponin T, were significantly higher in the 15-day culture groups than in the 10-day culture groups. Furthermore, the cardiac muscle-specific genes were expressed more in the high-serum groups compared to the low-serum groups regardless of the culture time. Cardiomyogenic differentiation of the P19 cells was most effective in 1 microM 5-azacytidine regardless of the serum concentrations. In addition, the stimulation effects of 5-azacytidine on cardiomyogenic differentiation were more significant under low-serum culture conditions compared to high-serum culture conditions. Cardiomyogenic differentiation of P19 cells was further confirmed by immunostaining with cardiac muscle-specific antibodies. CONCLUSION:Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells was enhanced by a combination of different experimental factors.
Actins ; Antibodies ; Azacitidine ; Carcinoma, Embryonal ; Cell Differentiation ; Dimethyl Sulfoxide ; Embryoid Bodies ; Embryonal Carcinoma Stem Cells ; Myocytes, Cardiac ; Real-Time Polymerase Chain Reaction ; RNA ; Safrole ; Troponin T ; Ventricular Myosins

BACKGROUND AND OBJECTIVES: The heat-shock response modulates contractility of vascular smooth muscles. With complementary deoxyribonucleic acid microarray, we tried to identify the novel genes that are involved in the regulation of vascular contraction after heat shock. MATERIALS AND METHODS: Human radial artery strips were mounted in organ baths, exposed at 42degrees C for 45 minutes, and returned to equilibrate at 37degrees C. This study examined gene expression profile associated with heat-shock response in radial arteries of patients with hyperlipidemia by using a microarray that contained 5763 human cDNA. The results of microarray hybridization experiments from the radial arteries of 4 different subjects were analyzed and classified by the cluster program. RESULTS: Among these differentially-expressed genes, Hsp70, Hsp10, alphaB-crystallin, and Hsp60 were significantly increased by the heat shock response. Of non-HSP genes, 15 genes increased, while 22 genes decreased. Among these 37 genes, alphaB-crystallin (CRYAB) (up 1.92-fold), myosin, light polypeptide kinase transcript variant 8, 6 (up 1.70-fold, up 1.68-fold), catenin (cadherin-associated protein, alpha-like 1) (down-0.57 fold) and tropomyosin 3 (down 0.68-fold) were thought to be related with the contraction. Real-time quantitative polymerase chain reaction showed that Hsp70, Hsp10 and alphaB-crystallin were significantly increased. CONCLUSION: Gene expression profile by heat shock provides information about genes implicated in augmentation of vascular contraction after heat shock.
Baths ; Chimera ; Contracts ; DNA ; DNA, Complementary ; Heat-Shock Response ; Hot Temperature ; Humans ; Hyperlipidemias ; Light ; Muscle, Smooth, Vascular ; Myosins ; Phosphotransferases ; Polymerase Chain Reaction ; Proteins ; Radial Artery ; Shock ; Transcriptome ; Tropomyosin

OBJECTIVETo explore the genetic basis and phenotypic correlation with disease severity in a large cohort of Chinese patients with hypertrophic cardiomyopathy (HCM).

METHODSA total of 179 unrelated Chinese HCM patients admitted to our department from 2002 to 2011 were enrolled in this study. Direct gene sequencing of β-myosin heavy chain (MYH7), myosin binding protein-C ( MYBPC3), and cardiac troponin T (TNNT2) were performed and clinical data were obtained in these patients.

RESULTSA total of 34 mutations were identified in 40 patients (22.3%), 79.4% (27/34) mutations occurred only once and a possible hot spot, A26 in MYH7, was found. Distribution of mutations was 52.9% (18/34) (MYBPC3), 35.3% (12/34) ( MYH7) and 11.8% (4/34) (TNNT2) respectively. Double mutations were identified in 2.2% (4/179) patients. Genotype-positive patients were associated with an earlier symptom onset, severer left ventricular hypertrophy, a higher incidence of syncope, and were more likely to have positive family history of HCM or sudden cardiac death (SCD) , and were more likely to progress into heart failure (24.2% vs. 5.0%, P = 0.002) and at a higher risk of SCD (9.1% vs. 0, P = 0.009) during the 6.5-year follow-up. No statistical difference in any clinical parameters and outcomes was found between patients carrying MYBPC3 and MYH7 mutations. Double mutations were associated with malignant clinical progression in this cohort. Different phenotype severity could be seen in HCM patients with same genotype (e. g. MYH7-1736T, TNNT2-R92W).

CONCLUSIONMYBPC3 is the most predominant gene mutation in this HCM cohort. The presence of a sarcomere mutation in patients with HCM is associated with poor clinical outcome, although no specific genes or mutations can exactly predict the severity of clinical phenotypes.

Asian Continental Ancestry Group ; Cardiomyopathy, Hypertrophic ; Carrier Proteins ; Death, Sudden, Cardiac ; Disease Progression ; Genotype ; Humans ; Hypertrophy, Left Ventricular ; Mutation ; Phenotype ; Sarcomeres ; Troponin T ; Ventricular Myosins

OBJECTIVETo test whether in the absence of actin, actin-binding proteins such as caldesmon, calponin, and tropomyosin interact with the myosin of unphosphorylation, Ca2+-dependent phosphorylation (CDP), and Ca2+-independent phosphorylation (CIP) and stimulate myosin Mg2+-ATPase activities.

METHODSMg2+-ATPase activities were measured to evaluate the effects of caldesmon, calponin, and tropomyosin on the myosin in unphosphorylation, CDP by myosin light chain kinase (MLCK), and CIP by MLCK.

RESULTS(1) At different incubation-time, i.e., 5, 10, 20, 40, and 60 minutes, the highest Mg2+-ATPase activity was observed when myosin was in the state of CDP, the medium was CIP of myosin, and the lowest was the unphosphorylated myosin. (2) In the absence of caldesmon, calponin, and tropomyosin, the Mg2+-ATPase activities from high to low were in the following order: CDP, CIP, and unphosphorylated myosin. However, in the presence of caldesmon, calponin, and tropomyosin, the order of relative value of Mg2+-ATPase activities from high to low was unphosphorylated, CIP, and CDP of myosin respectively compared to the corresponding controls.

CONCLUSIONSThe results propose that caldesmon, calponin, and tropomyosin are capable of stimulating Mg2+-ATPase activity of smooth muscle myosin in Ca2+-independent manner, since Ca2+ is not obligating for the stimulating effects of the three proteins. The common characteristic of the three proteins is that when myosin activities are low, their activations are relatively strong and this property might be involved in smooth muscle tension keeping.

Animals ; Ca(2+) Mg(2+)-ATPase ; drug effects ; metabolism ; Calcium ; pharmacology ; Calcium-Binding Proteins ; pharmacology ; Calmodulin-Binding Proteins ; pharmacology ; Chickens ; Microfilament Proteins ; Muscle, Smooth ; enzymology ; Myosins ; metabolism ; Phosphorylation ; Tropomyosin ; pharmacology

BACKGROUND AND OBJECTIVES: Spontaneous progression of severe congestive heart failure is structurally characterized by cellular degeneration and multiple foci of myocardial cell death. The cardiac troponin I (cTnI), one of the subunits of the troponin regulatory complex, binds to actin and inhibits interaction between actin and myosin. cTnI is uniquely expressed in the adult human myocardium, and an increase in its circulating levels is highly indicative of myocardial injury. In this study, we addressed the usefulness of cTnI as a sensitive and specific molecular marker for severity in patients with congestive heart failure. MethodscTnI, creatinin kinase-MB (CK-MB), and myoglobin were assessed in 59 patients with severe congestive heart failure diagnosed by the echo-cardiography and gated equilibrium blood pool heart scan. Also we assesed cTnI, creatinin kinase-MB (CK-MB), and myoglobin in 25 persons without cardiac disease in echocardiography. RESULTS: 1) The cTnI con-centration was 89.6+/-69.3 pg/mL in patients with congestive heart failure and its level was greater than that of the control group (22.4+/-17.1, p=0.001). 2) The cTnI level differed significantly according to left ventricular ejection fraction (EF), 117.3+/-73.8 pg/mL in patients with EF\<40% (28 patients), 66.3+/-44.5 pg/mL in patients with EF> or =40% (31 patients), 22.4+/-17.1 pg/mL in the control group (25 persons) (p=0.001). CONCLUSION: cTnI was useful as a specific and sensitive serum molecular marker in patients of congestive heart failure. And its level reflected the severity of congestive heart failure.
Actins ; Adult ; Cell Death ; Echocardiography ; Equidae ; Estrogens, Conjugated (USP)* ; Heart ; Heart Diseases ; Heart Failure* ; Humans ; Myocardium ; Myoglobin ; Myosins ; Stroke Volume ; Troponin I* ; Troponin*

PURPOSE: To identify novel proteins which bind to myocilin, using a yeast two-hybrid screening system. METHODS: A bait plasmid of myocilin was constructed, and then transformed into yeast AH109. The transformed yeast cells were mated with yeast Y187 containing human skeletal muscle cDNA library plasmids in 2 X YPD medium. Mated diploid yeasts were plated on synthetic dropout medium (SD/-Trp-Leu-His and SD/-Trp-Leu-His-Ade). The positive clones grown on the selective medium were amplified, sequenced, and analyzed using the database of GenBank. The expression of selected genes in trabecular meshwork (TM) cells was detected by RT-PCR. RESULTS: The expression of bait protein in yeast was confirmed by Western blot analysis. Extensive screening of a cDNA library led to the selection of twenty-four positive clones which represent eight genes, including six of cytoskeleton associated proteins such as alpha-actin, myosin regulatory light chain 2A, dynactin, syntrophin alpha1, microtubule associated protein 1B, and myosin binding protein C. CONCLUSIONS: Myocilin may interact with cytoskeleton associated proteins in TM cells. Further studies on their interactions will provide functional clues of myocilin.
Actins ; Blotting, Western ; Carrier Proteins ; Clone Cells ; Cytoskeleton ; Databases, Nucleic Acid ; Diploidy ; Gene Library ; Humans ; Mass Screening* ; Microtubules ; Muscle, Skeletal ; Myosin Light Chains ; Myosins ; Patient Dropouts ; Plasmids ; Trabecular Meshwork ; Yeasts

Actin and some actin binding proteins such as tropomyosin, -actinin and troponin T were localized by simultaneous double immunogold labeling in several developmental stages of Cryptosporidium parvum. All of the observed developmental stages have many particles of tropomyosin and actin around pellicle and cytoplasm. Tropomyosin was labeled much more than the actin when these two proteins were labeled simultaneously. And alpha-actinin was labeled mostly in the pellicle, but troponin T labeling was very rarely observed. From this study, it was suggested that tropomyosin seems to be one of the major proteins of C. parvum, so it must be playing important roles in C. parvum.
parasitology-protozoa ; actin ; tropomyosin ; alpha-actinin ; troponin T ; Cryptosporidium parvum

Carcinosarcoma of the female genital tract, also called malignant mixed mullerian tumor (MMMT), is a rare and relatively aggressive tumor with various homologous and heterologous components. There have been various studies to find prognostic factors and to investigate the histogenesis, including tissue culture, electron microscopy, and immunohistochemical studies. We investigated carcinomatous, sarcomatous, and transitional areas of 6 cases of carcinosarcoma of the uterus and ovary by using epithelial and mesenchymal markers. Immunohistochemical profiles of the transitional areas were significantly different from those of carcinomatous and sarcomatous areas. Immunoreactivities for cytokeratin and epithelial membrane antigen (50% and 22.2%) were weaker than those of carcinomatous areas (95.2% and 100%), but stronger than those of sarcomatous areas (11.1% and 5.6%)(p<0.01). In transitional areas, vimentin, smooth muscle actin and S-100 protein were more strongly expressed than in carcinomatous areas, but more weakly expressed than in sarcomatous areas (p<0.01, p<0.01, and p=0.018, respectively). Myoglobin was entirely negative in carcinomatous areas and immunoreactive in minor portions of transitional and sarcomatous areas (22.2% and 16.7%, respectively). These results suggest that the transitional areas are between the carcinomatous and sarcomatous nature in differentiation, further supporting that the carcinosarcomas of the female genital tract may arise, through metaplastic change, from a type of carcinoma.
Actins ; Carcinosarcoma* ; Female* ; Humans ; Immunohistochemistry ; Keratins ; Metaplasia ; Microscopy, Electron ; Mucin-1 ; Muscle, Smooth ; Myoglobin ; Ovary ; S100 Proteins ; Uterus ; Vimentin