第14回千葉カルシウム代謝研究会

(A) The DNA distribution of cells treated with bicyclol-AKT cDNA-, bicyclol-AKT cDNA+, bicyclol + AKT cDNA- and bicyclol + AKT cDNA+. The cDNA was transfected as mentioned in Methods. Then the cells were treated with bicyclol for 24 h (B) The DNA distribution of cells treated with bicyclol or/and LY294002 for 24 h. (C) The DNA distribution of cells treated with bicyclol or/and PD98059 for 24 h. (TIF 809 kb

Additional file 2: Table S2. of Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

Characteristics of ribosomally synthesized antimicrobial peptides of Bacillales. (DOCX 201 kb

Additional file 3: Table S3. of Identification and classification of known and putative antimicrobial compounds produced by a wide variety of Bacillales species

Characteristics of NRPs, PKs and NRPS/PKS hybrid synthesized antimicrobials of Bacillales. (DOCX 854 kb

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 Ca²⁺ 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

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

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

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

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