21.神経芽細胞腫肝転移例の1年生存(第589回千葉医学会例会・第2回千葉大学小児外科教室例会)

Additional file 2: Table S1. A list of phosphoPSMs identified by MASCOT and SEQUEST

CSC vapor decreased the expression of AK3 and increased cisplatin resistance.

<p>(a) Western blot analysis was performed using anti-<i>AK3</i> and anti-<i>AK2</i> antiserum. Protein extracts in each lane are as indicated. Even loading was confirmed by re-probing the membrane with β-Actin antibody. HUC1-6M or SCaBER-6M indicates the cells chronically exposed to CSC vapor. HUC1-0.1%6M are SV-HUC-1 cells exposed to 0.1% CSC for 6 months. SV-HUC-1 and HUC1-6M (b), SCaBER and SCaBER-6M (c) were treated with 0 to 5 µg/ml cisplatin for 48 h, or with 3 µg/ml cisplatin for 24, 48 and 72 h, in the presence and absence of AK3 as indicated. (d) Colony formation assays were performed with SCaBER and SCaBER-6M in the presence and absence of AK3 as indicated. Data were expressed as mean ±SD. * indicates <i>P</i><0.05 (e) SV-HUC-1 and SCaBER cells were transfected with AK3 RNAi for 48 h and Western blot analysis was performed using anti-AK3 antiserum. (f) SCaBER cells were transfected with AK3 RNAi and 48 h after transfection the cells were treated with 0 to 5 µg/ml cisplatin for 48 h and cellular survival was assessed. Error bars represent standard deviation of three experiments.</p

ΔΨm Alteration by AK3 in the Presence of Cisplatin.

<p>Mitochondrial membrane potential was measured in SV-HUC1, HUC1-6M (a), SCaBER and SCaBER-6M (b) cells transfected with AK3 or control vector, followed by treatment with cisplatin for 48 h. Cells were stained with JC-1 reagent and cell fluorescence was measured on a flow cytometer using Fl1 and Fl2 channels. Increase of red fluorescence indicates hyper-polarization of mitochondrial membrane potential (ÄΨm). * indicates <i>P</i><0.05 and ** indicates <i>P</i><0.01.</p

ROS Production by AK3 in the Presence of Cisplatin.

<p>ROS production was measured in parental SV-HUC-1 and SCaBER cells or cells exposed to CSC vapor for 6 months using DCFH-DA staining. The cells were transfected with or without the AK3 expression plasmid and treated with or without cisplatin for 48 h before staining. ROS generation in SV-HUC-1 and HUC1-6M (a–c) and SCaBER and SCaBER-6M (d–f) cells with overexpression of AK3 with and without cisplatin. The mean fluorescence density was calculated and data was plotted as mean ± SD (g,h).</p

AK3 Induced Release of Lactate Dehyderogenase upon Treatment of Cisplatin.

<p>LDH release in SV-HUC-1 (a) and SCaBER (b) cells exposed to CSC vapor in presence of AK3 followed by cisplatin treatment for 48 h. The data are presented as means ± SD. All experiments were performed in triplicate. Statistical significance is as indicated with * indicates <i>P</i><0.05 and ** indicates <i>P</i><0.01</p

Activation of Apoptosis in the Presence of AK3 and Cisplatin.

<p>Apoptosis was measured in the SV-HUC-1 and HUC1-6M cells using annexin/PI staining. The cells were transfected with or without AK3 expression plasmid and treated with or without cisplatin for 48 h as indicated.</p

Mycobacterial protein tyrosine kinase, PtkA phosphorylates PtpA at tyrosine residues and the mechanism is stalled by the novel series of inhibitors

<p>Phosphorylation and dephosphorylation are the key mechanisms for mycobacterial physiology and play critical roles in mycobacterial survival and in its pathogenesis. Mycobacteria evade host immune mechanism by inhibiting phagosome – lysosome fusion in which mycobacterial protein tyrosine phosphatase A (PtpA;TP) plays an indispensable role. Tyrosine kinase (PtkA;TK) activated by autophosphorylation; phosphorylates TP, which subsequently leads to increase in its phosphatase activity. The phosphorylated TP is secreted in phagosome of macrophage. In the present study, we have shown that the phosphorylation at two sites of TP; Y¹²⁸ and Y¹²⁹ are critical for TK-mediated phosphatase activity. The disruption of this interaction between TK and TP inhibits activation of later which further leads to the decrease in intracellular survival of mycobacteria. Furthermore, the proof of concept has been established using benzylbenzofurans and benzofuranamides, which inhibit the growth and intracellular survival of mycobacteria, associate with the functional sites of TP and contend with the TK. This binding was further restated by looking at the anchorage of protein–protein and the protein–inhibitor complexes in the homology-based structure models and by surface plasmon resonance analysis.</p

Effect of curcumin treatment on the <i>B</i>. <i>subtilis</i> AH75 growth and cell morphology.

<p><b>(A)</b><i>B</i>. <i>subtilis</i> AH75 strain was grown in LB media having spectinomycin antibiotic (100 μg/mL) till the OD₆₀₀ reached to 0.1. Then the cultures were treated with DMSO (control), 20 μM (IC₅₀ concentration) and 100 μM (MIC concentration) curcumin. Growth curve was plotted by measuring the OD₆₀₀ for all the samples at every 20 min interval till 360 min (mid-exponential phase). The three time points of curcumin treatment (20, 60 and 120 min) used in proteomic analysis are indicated by arrows. IC₅₀ concentration was used for subsequent proteomic analysis. (<b>B)</b><i>B</i>. <i>subtilis</i> AH75 strain was grown in the presence of 20 μM (IC₅₀ concentration) curcumin and the samples was collected after 20, 60 and 120 min of the drug treatment. Cultures treated with only DMSO was used as control. The nuclear materials were stained using 1 μg/μL DAPI for 20 min at room temperature in dark for all the samples. The fluorescence microscopic images were captured with both DAPI and DIC filters. The control <i>B</i>. <i>subtilis</i> cells showed normal cell length with one or two nucleoids per cell whereas after 20, 60 and 120 min of incubation with 20 μM (IC₅₀ concentration) curcumin, most of the cells turned into filamentous structure with multiple nucleoids. I- DIC image, II- DAPI image and III- overlay image.</p

(A) CTC staining and flow cytometric analysis for respiratory activity.

<p>Graphical representation of CTC mean intensity (PE-Texas Red-A) <i>vs</i>. FSC-A obtained in the FACS analysis of control, 20, 60 and 120 min curcumin treated samples and negative control. Both dot plot and histogram representations are displayed for each sample. <b>(B) & (C)</b> Potassium and phosphorus leakage assay; curcumin (20 and 40 μM) was added to the <i>B</i>. <i>subtilis</i> in HEPES-glucose medium and K⁺ and P levels were measured at 20, 60, 90 and 120 min time intervals, and also in the untreated control and positive control (heated at 70° C for 30 min) samples using ICP-AES and data was normalized with baseline HEPES-glucose medium. <b>(D)</b> Metabolic activity assay using resazurin. 20 min curcumin treatment has showed lower metabolic activity whereas the metabolic activity increased as time of exposure increased to 60 min and 120 min as compared to control. * indicates <i>p</i> < 0.05. <b>(E)</b> Gene expression analysis using RT-PCR for <i>murAA</i>, <i>spoVG</i> and <i>ftsH</i> genes and the relative expression was calculated by taking mean C_t values from triplicate runs. * indicates <i>p</i> < 0.05 and ** indicates <i>p</i> < 0.001. <b>(F)</b> Physical interaction analysis of curumin with <i>B</i>. <i>subtilis</i> FtsZ immobilized on CM-5 sensor chip. The interaction was monitored by measuring the response unit and the response unit was increased as the concentration of curcumin increased. Both sensorgram and the bar diagram showing the binding to FtsZ was displayed.</p

Quantitative profiles of the differentially expressed proteins involved in diverse biological processes identified in iTRAQ-based quantitative proteomics analysis using LTQ-Orbitrap.

<p><b>(A)</b> Peptidoglycan biosynthesis, <b>(B)</b> Fatty acid synthesis, <b>(C)</b> Cell division and sporulation, <b>(D)</b> TCA cycle, <b>(E)</b> Stress response and <b>(F)</b> Nucleotide biosynthesis. Data from QTOF is provided in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120620#pone.0120620.s002" target="_blank">S2 Fig</a>.</p