OBJECTIVE: We previously described that Diva is highly expressed in matured metaphase II (MII) oocytes compared to immature germinal vesicle (GV) oocytes in mouse.1 We report here that the expression of Diva transcript as well as protein is oocyte-specific. To elucidate its physiological role in oocyte, the binding partner(s) of Diva has been identified by using immunoprecipitation (IP) followed by Mass Spectrometry. METHODS: NIH/3T3 cells were transiently transfected for 24 h with either empty vector for control or FLAG-tagged mouse Diva construct, and IP was performed with anti-FLAG antibody. The immuno-isolated complexes were resolved by SDS-PAGE on a 12% gel followed by Coomassie Blue staining. For in-gel digestion, 15 bands of interest were excised manually and digested with trypsin. All mass spectra were acquired at a positive reflector mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Proteins were identified by searching the NCBI nonredundant database using MASCOT Peptide Mass Fingerprint software (Matrixscience, London). RESULTS: Diva-associated complexes were formed in FLAG-tagged mouse Diva-overexpressed NIH/3T3 cells via IP using anti-FLAG-conjugated beads. Among the excised 15 bands, actin and actin-binding proteins such as tropomyosin, tropomodulin 3, and alpha-actinin were identified. Binding between Diva and actin or tropomyosin was confirmed by IP followed by Western blot analysis. Both bindings were also detected endogenously in mouse ovaries, indicating that Diva works with actin and tropomyosin. CONCLUSIONS: This is the first report that immuno-isolated Diva-associated complexes are related to actin filament of the cytoskeletal system. When we consider the association of Diva with actin and tropomyosin, oocyte-specific Diva may play a role in modulating the cytoskeletal system during oocyte maturation.
Actin Cytoskeleton ; Actinin ; Actins ; Animals ; Blotting, Western ; Dermatoglyphics ; Digestion ; Electrophoresis, Polyacrylamide Gel ; Female ; Immunoprecipitation ; Mass Spectrometry* ; Metaphase ; Mice ; Microfilament Proteins ; Oocytes ; Ovary ; Proteomics ; Tropomodulin ; Tropomyosin ; Trypsin

Formins are widely distributed in eukaryotes such as fungi, plants and animals. They play crucial roles in regulating the polymerization of actin, coordinating the synergistic interactions between actin and microtubules, and determining cell growth and morphology. Unlike formins from fungi and animals, plant formins have been evolved into two plant-specific types. Generally, type Ⅱ formins are believed to regulate the polarized growth of cells, and type Ⅰ formins may regulate the cell expansion and division processes. Recent studies on the function of plant formins suggest it is inappropriate to classify the function of formins purely based on their structures. This review summarizes the domain organization of formins and their corresponding functions, as well as the underpinning mechanisms. Furthermore, the unsolved or unexplored issues along with future perspectives on plant formins are proposed and discussed.
Actins ; Formins ; Microfilament Proteins ; Plant Cells ; Plant Development ; Plants

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

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

The purpose of this study is to reveal the role of Girdin in regulating the aggregation of actin filaments by studying the relationship between PKB/Akt and Girdin. First we used Scansite software (http://scansite.mit.edu) to predict relevant target sites of PKB/Akt on mouse Girdin. To gain insight into the role of phosphorylation of Girdin by PKB/Akt, we assessed the location of phosphorylated Girdin in fertilized eggs by staining with anti-P-Girdin 1 417 Ab. We detected a distinct increase in the fluorescence signal of F-actin and P-Girdin 1 417 after microinjection of Akt WT and myr-Akt. The addition of myr-Akt induced phosphorylation of Girdin in mouse fertilized eggs. In addition, siRNA-mediated Akt-knockdown blocked phosphorylation of Girdin. The distribution of actin filaments was obviously scattered. These results strongly suggest that PKB/Akt could directly phosphorylate Girdin on Ser1 417 and promote its function in mouse fertilized eggs.
Actins ; physiology ; Animals ; Mice ; Microfilament Proteins ; physiology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; physiology ; RNA, Small Interfering ; Vesicular Transport Proteins ; physiology ; Zygote

AIMTo investigate the molecular mechanisms of smooth muscle 22 alpha (SM22alpha) whereby cytoskeleton remodeling of vascular smooth muscle cells (VSMCs) is regulated.

METHODSSynthetic (dedifferentiated) VSMCs were converted to contractile (differentiated) VSMCs by serum deprivation. Cells were transfected with pEGFP-SM22alpha, and localization of SM22alpha and its relationship with F-actin were observed through fluorescence microscopy. Fractional extraction of proteins and Western blotting were used to detect polymerization of SM alpha-actin in antisense-pcD2-SM22alpha-transfected VSMCs.Furthermore, effect of SM22alpha on F-actin cross-linking was observed by F-actin polymerization experiment.

RESULTSFluorescence microscopy showed that SM22alpha co-localized with F-actin in contractile VSMCs. Western blotting of protein extracts from F-/G-actin fractions revealed that polymerization of SM alpha-actin was lower in antisense-pcD2-SM22alpha-transfected VSMCs, in which SM alpha-actin mostly existed as soluble G-actin. Moreover, F-actin polymerization in vitro also showed that GST-SM22alpha could promote cross-linking of F-actin to form thick and bundled stress fibres,while extracts from VSMCs transfected with antisense-pcD2-SM22alpha could not effectively induce the polymerization of F-actin.

CONCLUSIONSM22alpha acts as a modulator to participate in VSMC cytoskeleton remodeling. It can not only induce polymerization of G-actin to F-actin, but also promote cross-linking of F-actin to bundled stress fibres, indicating its vital role in cytoskeleton remodeling.

Actins ; metabolism ; Animals ; Cytoskeleton ; metabolism ; Male ; Microfilament Proteins ; metabolism ; Muscle Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

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: 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

BACKGROUNDPelvic organ prolapse (POP) is a major health problem in adult women that involves many factors. No proteomic analysis has been conducted exclusively in POP patients. This study aimed to identify the differential expression of proteins that may be involved in POP by proteomic analysis.

METHODSSamples of the uterosacral ligament (USL) were collected from five POP patients and five non-POP patients matched according to age, parity, and menopausal status and analyzed using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the mRNA expression of proteins that showed differential expression in the proteomic analyses.

RESULTSProteins differentially expressed between POP and non-POP patients were detected. Eight proteins that were down-regulated in the POP group were identified by MALDI-TOF-MS. These proteins included electron transfer flavoprotein, apolipoprotein A-I, actin, transgelin, cofilin-1, cyclophilin A, myosin, and galectin-1, and their expression was verified by qRT-PCR.

CONCLUSIONUsing comparative proteomics, we identified eight differentially expressed proteins (including four cytoskeleton proteins and three proteins related to apoptosis) in the USL that may be involved in apoptosis associated with the tissue effects in POP pathophysiology.

Actins ; metabolism ; Aged ; Apolipoprotein A-I ; metabolism ; Cyclophilin A ; metabolism ; Cytoskeleton ; metabolism ; Female ; Flavoproteins ; metabolism ; Galectin 1 ; metabolism ; Humans ; Ligaments ; metabolism ; Microfilament Proteins ; metabolism ; Middle Aged ; Muscle Proteins ; metabolism ; Myosins ; metabolism ; Pelvic Organ Prolapse ; metabolism ; Postmenopause ; metabolism ; Proteomics ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Sacrum ; metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Uterus ; metabolism

To study the cellular changes and the potential role of CD2-associated protein (CD2AP) in podocyte differentiation, conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium under permissive condition at 33 °C. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to non-permissive condition at 37 °C. Simultaneously, untransfected cells were taken as differentiation control. The podocyte proliferation rate was determined by MTT method. The expressions of CD2AP, WT1, synaptopodin and nephrin mRNAs were examined by RT-PCR. CD2AP, WT1 and nephrin protein expressions were examined by Western blot. The distribution of CD2AP, nephrin, F-actin and tubulin in differentiated and undifferentiated podocytes was detected by laser scanning confocal microscopy. The results showed: (1) CD2AP, WT1 and nephrin were stably expressed in differentiated and undifferentiated podocytes while synaptopodin was only expressed in differentiated podocytes. (2) CD2AP and nephrin mRNA and protein expressions were up-regulated during podocyte differentiation (P<0.05). (3) CD2AP and tubulin were distributed in the cytoplasm and perinulcear region in undifferentiated podocytes, and F-actin was predominantly localized to a cortical belt and paralleled to the cell axis. Under differentiation condition, CD2AP distribution profile was presented as peripheral accumulation, tubulin took on fascicular style and F-actin extended into foot processes in podocytes. CD2AP colocalized with nephrin and F-actin in undifferentiated podocytes. (4) After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth was arrested; Synaptopodin, the differentiation podocyte marker, was apparently down-regulated; The differentiation of podocytes was delayed. The results demonstrate that podocyte differentiation is accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in podocyte differentiation.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; physiology ; Animals ; Cell Differentiation ; Cell Line ; Cytoskeletal Proteins ; physiology ; Cytoskeleton ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Podocytes ; cytology ; RNA, Small Interfering ; Transfection ; WT1 Proteins ; metabolism