Multiplex PCRを用いた簡便で感度の高い溺死診断法の開発

For diagnosing death due to drowning, the method of acid digestion of diatoms is widely used to detect plankton in the organs of the corpse. However, the method is limited by its　being complex, hazardous, time-consuming, and insufficiently sensitive. We therefore, developed a novel simple method to diagnose death due to drowning, and determined the location of drowning by detecting genes of representative bacteria in the environment. To procure all the information in one step, the multiplex PCR method was designed. For the diagnosis of drowning, the genes of upper respiratory indigenous bacteria, Streptococcus salivarius and Streptococcus sanguinis were used as indicators. For detection of the location of drowning, Aeromonas hydrophila and Microcystis aeruginosa were used as indicators of freshwater, and Vibrio harveyi as an indicator of seawater. A set of primers was designed for multiplex PCR. to amplify all the bacterial genes simultaneously. Using this method, 47 cases of drowning were examined, and the causes and locations of death were diagnosed.博士（医学）・乙第1428号・平成31年3月15

Subtle modulation of ongoing calcium dynamics in astrocytic microdomains by sensory inputs.

Astrocytes communicate with neurons through their processes. In vitro experiments have demonstrated that astrocytic processes exhibit calcium activity both spontaneously and in response to external stimuli; however, it has not been fully determined whether and how astrocytic subcellular domains respond to sensory input in vivo. We visualized the calcium signals in astrocytes in the primary visual cortex of awake, head‐fixed mice. Bias‐free analyses of two‐photon imaging data revealed that calcium activity prevailed in astrocytic subcellular domains, was coordinated with variable spot‐like patterns, and was dominantly spontaneous. Indeed, visual stimuli did not affect the frequency of calcium domain activity, but it increased the domain size, whereas tetrodotoxin reduced the sizes of spontaneous calcium domains and abolished their visual responses. The "evoked" domain activity exhibited no apparent orientation tuning and was distributed unevenly within the cell, constituting multiple active hotspots that were often also recruited in spontaneous activity. The hotspots existed dominantly in the somata and endfeet of astrocytes. Thus, the patterns of astrocytic calcium dynamics are intrinsically constrained and are subject to minor but significant modulation by sensory input

Natural history of medium-sized atrial septal defect in pediatric cases

AbstractBackgroundThe indication for surgical repair of atrial septal defect (ASD) is pulmonary to systemic blood flow ratio (Qp/Qs)>2.0, and therapeutic strategy depends on the facility in cases of Qp/Qs 1.5–2.0. Defect size increases with age, but hemodynamic changes of medium-sized ASD (Qp/Qs 1.5–2.0) are unknown.Methods and resultsFrom April 1, 1985 to March 31, 2008, we experienced 125 cases of cardiac catheterization for ASD. Twelve cases were re-evaluated without surgical repair. The first and second catheterizations were performed at median ages of 7 years (range, 2–13 years) and 16 years (range, 5–19 years), respectively. The mean follow-up period was 7 years. Qp/Qs increased from 1.6 to 2.0 during follow-up (p<0.05). Of four cases with Qp/Qs<1.5 at initial presentation, three had Qp/Qs≥1.5 at second inspection. Right ventricle diastolic volume (RVEDV/LVEDV) also increased.ConclusionsQp/Qs and RVEDV/LVEDV of medium-sized ASD increase together in childhood. Re-evaluation before adulthood should be considered in patients with no indications of ASD closure in childhood

Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development

International audienceThe transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner

Apoptosis-dependent externalization and involvement in apoptotic cell clearance of DmCaBP1, an endoplasmic reticulum protein of Drosophila

To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, we isolated proteins that bind to the extracellular region of Draper using affinity chromatography. One of those proteins has been identified to be an uncharacterized protein called Drosophila melanogaster calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Spontaneous remission of a non-small cell lung cancer possibly caused by anti-NY-ESO-1 immunity.

Spontaneous remission of malignant tumors is rare and the biological mechanism of such remission has not been addressed. We report the case of a 71-year-old Japanese patient with non-small cell lung cancer with a right hilar tumor and pleural dissemination that spontaneously regressed. NY-ESO-1 is a cancer/testis antigen that can elicit specific immune responses in patients with cancer. Strong anti-NY-ESO-1 immunity was detected in this patient. His tumor cells expressed NY-ESO-1 and MHC class I molecules. Anti-NY-ESO-1 immunity might have contributed to spontaneous remission in this patient