125 research outputs found
Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro
<div><p>Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca<sup>2+</sup> transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway.</p></div
Changes of ROCK related signaling molecules before or after the differentiation of ADSCs into cardiomyocytes.
<p>(A) Phosphorylation levels of JNK. (<b>B</b>) Phosphorylation level of Akt. Lane 1 represents undifferentiated ADSCs (day 3). Lanes 2 and 3 representbeating cells (day 16) in the absence or presence of Y-27632, respectively. Phosphorylation level  =  phosphorylation/total. * <i>P</i><0.05.</p
Immunofluorescence staining of contracting clone for cardiac- and muscle-proteins.
<p>Beating cells were with anti-cMHC (red), anti-α-actin (green), anti-cTnI (red) and anti-Nkx2.5 (red) antibodies. No specific staining was obtained with anti-MyoD (red) and anti-α-SMA (red) antibodies. Nuclei (blue) were stained with Hoechst 33342.</p
Differential protein expression by undifferentiated ADSCs and ADSCs-derived cells in the absence or presence of Y-27632.
<p>Expression of cMHC (A), cTnI (B), Connexin 45 (C) and RyR2/DHPR (D) in the ADSCs (day 3) and ADSCs-derived cells (day 16, in the absence or presence of Y-27632) by flow cytometer analyses. Right column figures represent results from 3 independent experiments. * <i>P</i><0.05.</p
Morphology and expression of cardiac markers before and after differentiation of ADSCs into cardiomyocytes.
<p>(A) Morphological change of ADSCs into cardiomyocytes in the absence (left panels) or presence (right panels) of ROCK-specific inhibitor Y-27632. After 6 days of culture, spindle shaped cells (black arrow), small round cells (gray arrow) and small tube cells (white arrow) appeared in the top panel, with no beating. After 12 days, single contractive cells (white arrow) were shown in the center panel; After 18dyas, myotube-like structure formed a cohesive network in the bottom panels and they began to contract synchronously. Scale bars  = 100 µm. (B) Expression of cardiac genes was determined by RT-PCR before (lane 1 for undifferentiated ADSCs) and after (lane 2 for beating cells) differentiation of ADSCs into cardiomyocytes. Rat heart tissue was used as a positive control (lane 4), with no cDNA as a negative control (lane 3). Expression of α-cardiac actin, α-MHC, β-MHC, ANP, cTnT, GATA4 and MEF-2C mRNA was higher in ADSCs-derived beating cells than in undifferentiated ADSCs.</p
Expression of cardiac proteins, excitation-contraction coupling proteins and signaling proteins in beating cardiomyocytes.
<p>Expression of MLC-2v(A), cTnT(B) and RyR2/DHPR (C) in the SCR siRNA and ROCK siRNA groups by flow cytometer analyses. (D) Change of JNK and PI3K/Akt from Western Blot analyses. Lane 1 represents undifferentiated ADSCs (day 3), while lanes 2 to 5represent SCR siRNA, ROCKI siRNA, ROCKII siRNA and Both siRNA groups, respectively (day 16). Phosphorylation level  =  phosphorylation/total. * <i>P</i><0.05.</p
Primers for reverse transcription PCR and real time PCR and sequences for RNA interference.
<p>Primers for reverse transcription PCR and real time PCR and sequences for RNA interference.</p
Morphological change of actin cytoskeleton during the differentiation of ADSCs.
<p>ADSCs and ADSCs-derived cardiomyocytes were stained with phalloidin for F-actin (green). The white arrows point to stained stress fibers. After 3 days of culture in the absence of Y-27632 (top left panel), stress fibers in cells weres parse. After 7 days of culture in the absence of Y-27632 (top right panel), stress fibersshifted from sparse to dense distribution. The long stress fibers fully occupied the cytoplasm of contractive cells after 16 days of culture in the absence of Y-27632 (middle left and bottom left panels). The contractive cells in the presence of Y-27632 showed much less compact stress fibers in the cytoplasm, in comparison with un-treated cells (after 16 days of culture, middle rightand bottom right panels). The white arrow indicates stress fibers. Nuclei (blue) were stained with Hoechst 33342, Scale bar  = 100 µm.</p
Dirhodium(II)-Catalyzed Sulfide Oxygenations: Catalyst Removal by Coprecipitation with Sulfoxides
The
dirhodiumÂ(II) carboxylate complex Rh<sub>2</sub>(esp)<sub>2</sub> (esp
= α,α,α′,α′-tetramethyl-1,3-benzenedipropanoate)
was shown to catalyze the sulfoxidation of organic sulfides using <i>tert</i>-butyl hydroperoxide as the oxidant. Due to the unique
structure of Rh<sub>2</sub>(esp)<sub>2</sub> and its stable Rh<sub>2</sub>(II,II) catalyst resting state, the rhodium catalyst is able
to precipitate as a Rh<sub>2</sub>(esp)<sub>2</sub>–sulfoxide
complex following the reaction which makes separation of the catalyst
from the products very convenient. The precipitated Rh<sub>2</sub>(esp)<sub>2</sub>–sulfoxide complexes could be reused to catalyze
sulfide oxygenation reactions without considerable loss of activity.
Mechanistic studies suggest that the axial ligands fine-tune the redox
potential of the dirhodiumÂ(II,II) compounds and determine the predominant
catalyst species in the oxidation reaction
TiO<sub>2</sub>‑Catalyzed <i>n</i>‑Valeraldehyde Self-Condensation to 2‑Propyl-2-Heptenal: Acid Catalysis or Base Catalysis?
Several TiO<sub>2</sub> samples with
different morphologies and structures, nano/microcomposite of TiO<sub>2</sub> (anatase), TiO<sub>2</sub> whisker (anatase), and nano-TiO<sub>2</sub> (anatase), were prepared and characterized by means of N<sub>2</sub> adsorption–desorption, CO<sub>2</sub>-TPD, and NH<sub>3</sub>-TPD, and their catalytic performance for <i>n</i>-valeraldehyde self-condensation was investigated. The results indicated
that the conversion of <i>n</i>-valeraldehyde was correlated
with their acid amount while the selectivity of 2-propyl-2-heptenal
was associated with their base amount. Since the catalytic performance
of nano-TiO<sub>2</sub> (anatase) was the best, its preparation process
was further studied and the suitable preparation conditions were obtained.
Then the effect of reaction conditions on the catalytic performance
of nano-TiO<sub>2</sub> (anatase) for <i>n</i>-valeraldehyde
self-condensation was investigated and the suitable reaction conditions
were obtained as follows: a weight percentage of TiO<sub>2</sub> catalyst
of 15 wt %, a reaction temperature of 190 °C, and a reaction
time of 10 h. Under the above reaction conditions, the conversion
of <i>n</i>-valeraldehyde, 2-propyl-2-heptenal yield, and
selectivity were 94.6%, 93.7%, and 99.1%, respectively. The TiO<sub>2</sub> catalyst could be reused four times without a significant
loss in its catalytic performance, which was different from most of
the literature. The catalytic stability of TiO<sub>2</sub> catalyst
was associated with the properties of the active sites, especially
acid–base property. Not as some of the literature claimed that
their TiO<sub>2</sub>-catalyzed reactions were base-catalyzed reactions,
the TiO<sub>2</sub> catalyst used in this work possessed much greater
acid amount than base amount. To assess the role of acidic and basic
sites in <i>n</i>-valeraldehyde self-condensation, ammonia
and carbon dioxide were separately used as a probe molecule for poisoning
the corresponding active sites. The results confirmed the key role
of acid sites in <i>n</i>-valeraldehyde self-condensation.
Therefore, we were convinced that the TiO<sub>2</sub>-catalyzed <i>n</i>-valeraldehyde self-condensation was mainly an acid-catalyzed
reaction
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