Thirteen-week Intravenous Toxicity Study of a Novel Humanized Anti-Human Death Receptor 5 Monoclonal Antibody, CS-1008, in Cynomolgus Monkeys

CS-1008, a humanized monoclonal antibody that is agonistic to human death receptor 5, was intravenously administered to cynomolgus monkeys twice a week for 13 weeks at 3 different dose levels (5, 15 and 42 mg/kg) in order to evaluate its potential toxicity. A control group received phosphate buffered saline containing 0.01% polysorbate 80. Each of the 4 groups consisted of 3 male and 3 female cynomolgus monkeys. No animal in any group died during the dosing period. No toxic changes in clinical signs, food consumption, body weight, electrocardiography, ophthalmology, urinalysis, hematology, blood chemistry, gross pathology, organ weights or histopathology were noted in any group during the dosing period. In the toxicokinetic analysis, the values for the maximum concentration of CS-1008 in plasma and the area under the curve generally increased with increasing dose. No clear differences in the toxicokinetic parameters or profiles were observed between the sexes. Development of anti-CS-1008 antibodies was not detected in any sample. The no-observed adverse-effect level (NOAEL) of CS-1008 in cynomolgus monkeys under the conditions of this study was concluded to be 42 mg/kg in both sexes, when administered intravenously twice a week for 13 weeks. This study supports the development of CS-1008 as a therapeutic biopharmaceutical

保育現場が求める実習生像の分析

本研究の目的は、保育現場が求める実習生像を明らかにすることである。実習生に身につけておいてほしいことがらを尋ねる26項目を作成し、保育者214名に質問紙調査を実施した。保育現場が求める実習生像について、幼稚園と保育所および公立と私立という勤務先による特徴を検討した。さらに、保育現場での立場として、経験年数、職場内の立場、実習生の年間の受入人数を取りあげて、保育現場が求める実習生像との関係を検討した。分析の結果、次の３点が示された。第１に、保育現場が実習生に求めることは、「学ぶ姿勢・態度」と「保育実践のスキル」にまとめることができる。第２に、「学ぶ姿勢・態度」は、私立施設勤務者が公立施設勤務者よりも実習生に必要であると考えていた。第３に、「学ぶ姿勢・態度」は、園長、主任、保育士が幼稚園教諭よりも実習生に必要であると考えていた。以上の結果を踏まえて、実習指導および保育者養成について、「学ぶ姿勢・態度」を育てることを通して「保育実践のスキル」を身につけることができる指導が重要であること、保育現場が求める実習生像を実習生自身が知ることで実習への意識を高めること、実習先の保育現場が求める実習生像を理解した上で実習生に指導を行なうことを提起した

In Vivo Imaging of Hierarchical Spatiotemporal Activation of Caspase-8 during Apoptosis.

[Background]: Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. [Methodology/Principal Findings]: We have established a biosensor based on fluorescence resonance energy transfer (FRET) for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET) system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3) in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. [Conclusions]: Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8

Dammarane-type triterpene extracts of <i>Panax notoginseng</i> root ameliorates hyperglycemia and insulin sensitivity by enhancing glucose uptake in skeletal muscle

<p>Skeletal muscle is an important organ for controlling the development of type 2 diabetes. We discovered <i>Panax notoginseng</i> roots as a candidate to improve hyperglycemia through <i>in vitro</i> muscle cells screening test. Saponins are considered as the active ingredients of ginseng. However, in the body, saponins are converted to dammarane-type triterpenes, which may account for the anti-hyperglycemic activity. We developed a method for producing a dammarane-type triterpene extract (DTE) from <i>Panax notoginseng</i> roots and investigated the extract’s potential anti-hyperglycemic activity. We found that DTE had stronger suppressive activity on blood glucose levels than the saponin extract (SE) did in KK-<i>A</i>^<i>y</i> mice. Additionally, DTE improved oral glucose tolerance, insulin sensitivity, glucose uptake, and Akt phosphorylation in skeletal muscle. These results suggest that DTE is a promising agent for controlling hyperglycemia by enhancing glucose uptake in skeletal muscle.</p> <p>DTM improved hyperglycemia.</p

A mathematical model on the propagation of CASP8 activation.

<p>(<b>A</b>–<b>C</b>) The propagation of CASP8 activation was simulated by an one-dimensional diffusion model. The vertical axis (<i>u</i>) indicates the concentration of the activated CASP8. The horizontal axis (<i>x</i>) indicates the distance from the input source of the apoptotic signal and time zero means the starting point of the cell-cell interaction. CASP8 activation propagates from the left to the right in the graph. Each colored line in the figures gives the spatial distribution of activated CASP8 for different time, where time zero is the starting point of the cell-cell interaction. A blue line indicates the start time when the apoptotic signal is inputted. A yellow line indicates 2000 unit time that has passed from the start time. The duration (<i>τ</i>) of the input signal and was set to 100 (A), 500 (B), and 1000 (C) and <i>f</i>₀ = 10. Here, diffusion coefficient is set to <i>D</i> = 1 and the cell size is set to <i>L</i> = 200.</p

Monitoring of caspase activation with the CYR83 in single cells.

<p>(<b>A</b>) A schematic structure of CYR83 and its variants. In CYR83(IETA) variant, the IETD sequence was replaced by IETA; in the CYR83(DEVA) variant, DEVD was replaced by DEVA. (<b>B</b>) The graphic pattern of the emission ratio based on the fluorescence intensity of the CYR83 in single cells undergoing apoptosis. The CYR83-expressing HeLa cells were induced to undergo apoptosis with an agonistic anti-Fas antibody and monitored by dual-FRET. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050218#pone.0050218.s003" target="_blank">Figure S3</a>, time course was set up before and after 1 h of cell shrinkage. The temporal fluctuations of the emission ratio on Venus/seCFP and mRFP1/Venus in single cells are plotted as red and blue lines, respectively. The IETDase and DEVDase activities are inversely proportional to graphic data. The arrow indicates a rebound detected by monitoring the fluorescence. (<b>C</b>) The CYR83-expressing HeLa cells were induced to undergo apoptosis by UV-irradiation and monitored by dual-FRET. A time course of the emission ratio is indicated. (<b>D</b>, <b>E</b>) HeLa cells expressing CYR83 variants were monitored for fluorescence. Transfected cells expressing CYR83(IETA) (D) or CYR83(DEVA) (E) were treated with an anti-Fas antibody and monitored for fluorescence. <b>(F</b>, <b>G)</b> Fluorescence image analyses on the proteolytic processing profiles of CYR83 and its variants. HeLa cells expressing CYR83 or its variants were subjected to extrinsic (F) and intrinsic (G) apoptotic stimuli at indicated times. Cell extracts prepared from those cells were resolved by SDS-PAGE and scanned for fluorescence in the gel using the imaging analyzer. Among fluorescence bands detected in panels of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050218#pone.0050218.s005" target="_blank">Figure S5A–C and S5E–G</a>, three bands corresponding to each seCFP, Venus and mRFP1 peptide fragments were chosen and represented as (F) and (G).</p