Electro-optic Bdot probe measurement of magnetic fluctuations in plasma

0000-0001-8457-5570We propose a combined use of a Pockels electro-optic sensor with a pickup loop coil (Bdot probe) for the measurement of magnetic fluctuations in plasmas. In this method, induced fluctuating voltage on the coil loop is converted into an optical signal by a compact electro-optic sensor in the vicinity of the measurement point and is transferred across optical fiber that is unaffected by electric noise or capacitive load issues. Compared with conventional Bdot probes, the electro-optic Bdot probe (1) is electrically isolated and free from noise pickup caused by the metallic transmission line and (2) can be operated at a higher-frequency range because of the smaller capacitance of the operation circuit, both of which are suitable for many plasma experiments. Conversely, the sensitivity of the current electro-optic Bdot probe arrangement is still significantly lower than that of conventional Bdot probes. A preliminary measurement result with the electro-optic Bdot probe showed the detection of a magnetic fluctuation signal around the cyclotron frequency range in the RT-1 magnetospheric plasma experiment.journal articl

B16 cells induced platelet aggregation in a cell number and P2Y12-dependent manner.

<p>(<b>A</b>) In order to examine the ability of B16 melanoma cells to directly cause platelet aggregation in vitro, 300μl suspensions containing 2×10⁵/ml, 6×10⁵/ml or 1×10⁶/ml of Ds-Red labeled B16 cells, respectively and 3×10⁸/ml calcein–labeled WT or P2Y12^−/− platelets were prepared, and aggregation was monitored using a Chrono-Log aggregometer. B16 melanoma cells were able to induce visible platelets-cancer cells aggregates. The aggregation was dependent on cancer cell number and WT platelets, and consequently was not supported by P2Y12 deficient platelets (n=3). (<b>B</b> & <b>C</b>) In order to detect the direct interaction between the B16 melanoma cells and the platelets in vitro, after the 20 minute aggregation trial, aliquots of each mixture of B16 cells and platelets were smeared on glass slides, and bright-field and fluorescent images were recorded at 10× and 20× magnifications. The images clearly revealed that some of the B16 cells directly interacted with WT platelets resulting in platelet accumulation on the surface of the B16 cells. In contrast, the P2Y12 deficient platelets did not support extensive accumulation of platelets on the surface of B16 cells (n=3).</p

LLC cell induced platelet shape change is P2Y12-dependent.

<p>(<b>A</b>) In order to investigate the direct interaction between LLC tumor cells and platelets in vitro, 2×10⁵ /ml, 6×10⁵ /ml or 1×10⁶ /ml of Ds-Red labeled LLC cells were added to a suspension of 3×10⁸/ml calcein–labeled WT or P2Y12^−/− platelets in total volume of 300μl, and aggregation and shape change were monitored for 20 minutes with stirring at 1000rpm using a Chrono-Log aggregometeras described. In contrast to the WT platelets, P2Y12 deficient platelets did not undergo shape change or aggregate in response to stirring in the presence of LLC cancer cells (n=3). (<b>B</b>) After stirring, a sample of the mixture of LLC cells and platelets was smeared on the slides, and bright-field and fluorescence images of were taken in the 10x objective field. No obvious direct interaction between LLC cells and platelets was observed.</p

Platelets promote invasiveness of LLC cells by inducing a P2Y12 and TGF-β1 dependent epithelial-mesenchymal-like transition (EMLT).

<p>(<b>A</b>) Platelet P2Y12 regulated LLC cell invasion in matrigel. As evident from the images and bar graphs, P2Y12 deficient platelets significantly inhibited LLC cell invasiveness. LLC cells were added at the top of transwells coated with matrigel and those cells were treated with buffer, WT platelets and P2Y12^−/− platelets, respectively. The effects of this treatment were evaluated by counting the crystal violet stained cells at the bottom of the wells after 40 hours incubation with buffer or platelets. Each bar represents the mean number of invasive cells ± SEM, statistical significance was determined using a one-way AVONA (10 random fields per sample, n=3 samples, ***p<0.001). (<b>B</b>) LLC cells induced the release of active TGF-β1 from platelets. The LLC cells attached to each well were incubated with buffer, WT or P2Y12^−/− platelets for 48 hours. Cancer cells and platelets were removed from the conditioned medium by centrifugation. The amounts of the active form of TGF-β1 in the supernatant fractions were measured using ELISAs. Each bar represents the mean ± SEM, and the statistical significance was calculated using a one-way AVONA (***p<0.001, n=3). (<b>C</b>) The levels of active TGF-β1 were measured in the supernatant fractions from 300μl volumes of washed platelets stimulated by 20μM ADP, 2μg/ml collagen or 0.1U/ml α-thrombin. EDTA (final concentration of 5mM) was added to each platelet suspension before the supernatant fractions were prepared by centrifugation. The level of TGF-β1 was measured in each supernatant fraction using an ELISA. Each bar represents the mean ± SEM, and the statistical significance was calculated using a one-way AVONA (***p<0.001, n=3). (<b>D</b>) 10× and 20× phase-contrast micrographs of LLC cells that had been incubated with buffer, WT platelets, P2Y12^−/− platelets or 20ng/ml recombinant active TGF-β1 (as a positive control). In contrast to the P2Y12 deficient platelets, incubation of the LLC cells with WT platelets or active TGF-β1 obviously induced the LLC cells to undergo an EMT-like transition.</p

P2Y12 deficiency decreases lung metastasis but has no effect on primary tumor growth in the LLC (Lewis lung carcinoma) spontaneous pulmonary metastasis model.

<p>Each group of 9 mice was injected intradermally with 2×10⁶ LLC cells. Subcutaneous primary tumors were removed from each mouse and weighed 14 days after implantation. One month after removal of the subcutaneous tumors, each mouse was dissected, and its lungs were removed, weighed, and photographed. (<b>A</b>) Representative images of gross metastatic lungs from each group of mice. (<b>B</b>) Representative images of tissue sections from metastatic lungs obtained from WT and P2Y12 deficient mice. The lungs were paraffin-embedded prior to sectioning. The sections were stained with hematoxylin and eosin. (<b>C</b>) The mean weights of the primary tumors from the WT and P2Y12^−/− groups. Each bar represents the mean ± SEM of the weights of all the subcutaneous tumors from each group of mice. There was no statistically significant difference between the mean tumor weights of the WT and P2Y12 deficient groups (n=9). (<b>D</b>) The mean weights of metastatic lungs from WT and P2Y12^−/− groups. Each bar represents the mean ± SEM. P2Y12 deficiency significantly inhibited pulmonary metastasis (*p<0.05, n=9).</p

P2Y12 regulates/affects the formation of the pre-metastatic microenvironment in the lungs.

<p>(<b>A</b>) Representative images of fibronectin expression in lung sections. The levels of tissue fibronectin were measured in lung sections prepared at 3 and 14 days after intradermal implantation of the LLC cells. Immunostaining analysis was used to measure tissue fibronectin. The levels of lung tissue fibronectin were obviously enhanced at day 14 in the WT group, but not in the P2Y12 deficient group. In the latter group, fibronectin was expressed mainly in the regions of micro-vessels and terminal bronchi. Arrows indicate the fibronectin positive areas in the lungs of each group, and enlarged insets in 40× magnification are shown. (<b>B</b>) Statistical analyses of the percentages of fibronectin positive areas in sections of the total lungs for all the animals in each group. The lungs from both groups of animals were approximately the same size. P2Y12 deficiency did not support metastasis-driven enhancement of fibronectin expression (3 sections for each mouse; n=5 for each group, *p<0.05). (<b>C</b>) The numbers of VEGFR1 positive cells were measured in lung sections prepared on days 3 and 14 after intradermal injection of the LLC cells. By day 14, VEGFR1^<b>+</b> cell clusters were apparent surrounding the distal bronchials and microvascular regions of the WT mouse lungs. In contrast, the P2Y12 deficient mice had few VEGFR1+ cell clusters in their lungs. Arrows indicate the VEGFR1^<b>+</b> cell clusters in the lungs of each group, and enlarged 40× insets in are shown. (<b>D</b>) Statistical analyses of the mean number of VEGFR1^<b>+</b> cells per lung section P2Y12 deficiency did not support metastasis-driven recruitment of VEGFR1^<b>+</b> cells to the lungs. Each bar represents the mean ± SEM, and statistical significance was determined using a one-way AVONA (3 sections for each mouse; n=5 for each group, *p<0.05).</p

P2Y12 facilitates a TGF-β1-independent EMT-like transition of B16 cells, and experimental pulmonary metastasis by B16 cells.

<p>(<b>A</b>) Phase-contrast 10× and 20× micrographs of B16 tumor cells incubated with buffer, WT platelets, P2Y12^−/− platelets or 20ng/ml active TGF-β1, respectively for 48 hours. Incubation with WT platelets obviously induced B16 cells to undergo an EMT-like transition, P2Y12 deficient platelets did not induce an EMT-like transition of B16 cells. Interestingly, the recombinant active TGF-β1 induced the B16 cells to undergo a less extensive EMT-like morphologic change than was induced by the WT platelets. (<b>B</b>) The level of active TGF-β1 in the B16 conditioned medium was also measured by an ELISA. B16 cells induced significantly less release of active TGF-β1 from P2Y12 deficient platelets than from WT platelets. Each bar represents the mean ± SEM, and statistical significance was determined using a one-way AVONA (**p<0.01, n=3). (<b>C</b>) Each mouse from both groups (n=7 for each group) were injected with 2 × 10⁵ B16 cells via a tail vein. Twenty days after injection of the B16 cells, the, lungs were dissected from each mouse, and photographed. Images of visible metastatic foci are apparent in the photomicrographs. (<b>D</b>) Statistical analyses of the number of metastatic foci at the surface of the lung lobes. Each bar represents the mean ± SEM, and statistical significance was determined using the Student’s t test (**p<0.01, n=7 for each group). (<b>E</b>) Representative histochemical images (10×) of lung sections from wild-type and P2Y12 deficient mice.</p

13) 早期十二指腸癌の1例(I.一般演題, 第44回新潟癌治療研究会）

departmental bulletin pape

Integrin αIIb-Mediated PI3K/Akt Activation in Platelets

<div><p>Integrin αIIbβ3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the β3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the β3 cytoplasmic domain residues R₇₂₄KEFAKFEEER₇₃₄. In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by β3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated β3 peptide R₇₂₄KEFAKFEEER₇₃₄, each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbβ3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbβ3-Δ724 or αIIbβ3E₇₂₄AERKFERKFE₇₃₄, but not in cells expressing wild type αIIbβ3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the β3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.</p> </div

SFK(s) and PI3K propagate αIIb-initiated signaling that elicits platelet activation, TxA2 production and granule secretion.

<p>Normal human platelets and β3Δ724 platelets were stimulated by the LIBS-specific monoclonal antibody D3 (30 µg/ml) in the presence of Fg (250 µg/ml) with or without 10 µg/ml of human αIIbβ3 specific monoclonal antibody 7E3, DMSO, 10 U/ml of apyrase, 75 µM of indomethacin, 10 µM of Src family kinase inhibitor PP2, 100 nM of PI3K inhibitor wortmannin, or 10 µM of MAPK inhibitor U0126. (<b>A</b>) The agglutination and/or aggregation of normal human platelets (NP) and β3Δ724 human platelets, respectively induced by D3 plus Fg with or without inhibitors treatment. The absence of shape change indicates agglutination, rather than aggregation. (<b>B</b>) The TxB2 production and granule secretion of normal human platelets and β3Δ724 platelets induced by D3 plus Fg with or without inhibitor treatment. Each bar represents the mean of quadruplicate determinations. The error bars correspond to the standard deviations of the data.</p