Quantitative evaluation of malignant gliomas damage induced by photoactivation of IR700 dye.

The processes involved in malignant gliomas damage were quantitatively evaluated by microscopy. The near-infrared fluorescent dye IR700 that is conjugated to an anti-CD133 antibody (IR700-CD133) specifically targets malignant gliomas (U87MG) and stem cells (BT142) and is endocytosed into the cells. The gliomas are then photodamaged by the release of reactive oxygen species (ROS) and the heat induced by illumination of IR700 by a red laser, and the motility of the vesicles within these cells is altered as a result of cellular damage. To investigate these changes in motility, we developed a new method that measures fluctuations in the intensity of phase-contrast images obtained from small areas within cells. The intensity fluctuation in U87MG cells gradually decreased as cell damage progressed, whereas the fluctuation in BT142 cells increased. The endocytosed IR700 dye was co-localized in acidic organelles such as endosomes and lysosomes. The pH in U87MG cells, as monitored by a pH indicator, was decreased and then gradually increased by the illumination of IR700, while the pH in BT142 cells increased monotonically. In these experiments, the processes of cell damage were quantitatively evaluated according to the motility of vesicles and changes in pH.

Arachidonic acid-dependent carbon-eight volatile synthesis from wounded liverwort (Marchantia polymorpha).

Eight-carbon (C8) volatiles, such as 1-octen-3-ol, octan-3-one, and octan-3-ol, are ubiquitously found among fungi and bryophytes. In this study, it was found that the thalli of the common liverwort Marchantia polymorpha, a model plant species, emitted high amounts of C8 volatiles mainly consisting of (R)-1-octen-3-ol and octan-3-one upon mechanical wounding. The induction of emission took place within 40min. In intact thalli, 1-octen-3-yl acetate was the predominant C8 volatile while tissue disruption resulted in conversion of the acetate to 1-octen-3-ol. This conversion was carried out by an esterase showing stereospecificity to (R)-1-octen-3-yl acetate. From the transgenic line of M. polymorpha (des6(KO)) lacking arachidonic acid and eicosapentaenoic acid, formation of C8 volatiles was only minimally observed, which indicated that arachidonic and/or eicosapentaenoic acids were essential to form C8 volatiles in M. polymorpha. When des6(KO) thalli were exposed to the vapor of 1-octen-3-ol, they absorbed the alcohol and converted it into 1-octen-3-yl acetate and octan-3-one. Therefore, this implied that 1-octen-3-ol was the primary C8 product formed from arachidonic acid, and further metabolism involving acetylation and oxidoreduction occurred to diversify the C8 products. Octan-3-one was only minimally formed from completely disrupted thalli, while it was formed as the most abundant product in partially disrupted thalli. Therefore, it is assumed that the remaining intact tissues were involved in the conversion of 1-octen-3-ol to octan-3-one in the partially disrupted thalli. The conversion was partly promoted by addition of NAD(P)H into the completely disrupted tissues, suggesting an NAD(P)H-dependent oxidoreductase was involved in the conversion.

A non-invasive imaging for the in vivo tracking of high-speed vesicle transport in mouse neutrophils.

Neutrophils play an essential role in the innate immune response. To understand neutrophil activity, the development of a new technique to observe neutrophils in situ is required. Here, we report the development of a non-invasive technique for the in vivo imaging of neutrophils labeled with quantum dots, up to 100 µm below the skin surface of mice. Upon inflammation neutrophils began to extravasate from blood vessels and locomoted in interstitial space. Most intriguingly, the quantum dots were endocytosed into vesicles in the neutrophils, allowing us to track the vesicles at 12.5 msec/frame with 15-24 nm accuracy. The vesicles containing quantum dots moved as "diffuse-and-go" manner and were transported at higher speed than the in vitro velocity of a molecular motor such as kinesin or dynein. This is the first report in which non-invasive techniques have been used to visualize the internal dynamics of neutrophils.

The generation of pancreatic ß-cell spheroids in a simulated microgravity culture system.

Islet transplantation can induce a substantial improvement in the treatment of type 1 diabetes mellitus. However, the clinical application of islet transplantation is severely limited by the shortage of donor organs. It is thus essential to improve the engraftment rate to achieve the expected outcome in the treatment of diabetes mellitus using a limited amount of donor islets. In this manuscript, we describe the generation of ß-cell spheroids using mouse insulinoma cells (MIN6) as a model of ß-cells. We established a 3D culture system that simulates microgravity using a 3D clinostat. Using this method, we were able to produce 100 spheroids per mL of culture media. The optimization of the culture conditions in the clinostat produced spheroids with a size of approximately 250 µm, which is a size that is known to induce good graft survival after islet transplantation. The spheroids produced in the clinostat expressed several ß-cell signature genes at higher levels than the levels that were found in MIN6 cells that were cultured in a standard 2D culture dish (MIN6-2D). The transplantation of the spheroids into the portal vein of streptozotocin-induced diabetic mice ameliorates hyperglycemia, whereas the transplantation of the equivalent number of 2D-cultured cells failed to cure diabetes. These results indicate that the clinostat culture provides a new method for the reconstitution of a large number of functional ß-cell spheroids for diabetes treatment.

Real-time and noninvasive monitoring of respiration activity of fertilized ova using semiconductor-based biosensing devices.

In this report, we propose a novel evaluation method of embryo activity, describing the real-time and noninvasive electrical monitoring of embryo activity, caused by fertilization of the sea urchin, using a biologically-coupled field-effect transistor (bio-FET) comprised of semiconductor-based biosensing devices. The detection principle of bio-FET is based on the potentiometric detection of charge density change at the gate insulator, which includes changes of hydrogen ion concentration corresponding to pH variation. The surface potential at the gate surface of the bio-FET increased after the introduction of sperms into the ova, resulting in fertilization on the gate sensing area. The positive shift of surface potential indicates the increase of positive charges of hydrogen ions generated by dissolved carbon dioxide in artificial sea water based on respiration activity of the embryo. Moreover, the electrical signal of embryo activity is suppressed due to the inhibition of cytokinesis by introduction of cytochalasin B. The platform based on the bio-FET is expected to be a real-time, label-free and noninvasive detection system, not only in fundamental studies of embryo activity but also in the evaluation of embryo quality for in vitro fertilization.

Dose-dependent effects of hypergravity on body mass in mature rats.

INTRODUCTION: Previous reports have shown that exposure to hypergravity decreases rat body mass during the initial phase, with this decrease and level of gravity showing a dose-response relationship. The present study examined whether rate of body mass gain after the initial phase of exposure is attenuated by hypergravity in a dose-dependent manner and sought to identify any threshold. METHODS: Male 10-wk-old rats (n = 64) were used, with 16 rats serving as 1.0-G controls, and 48 rats exposed to hypergravity for 14 d in 4 groups (1.5, 2.0, 2.5, and 3.5 G; n = 12 each). Body mass gain was evaluated according to slope of change in body mass from day 7 of exposure to hypergravity, as both absolute and relative values. RESULTS: Slopes of body mass gain did not differ between the 1.0- and 1.5-G groups (6.09 and 5.75 g x d(-1), respectively), but were significantly less for the 2.0-, 2.5-, and 3.5-G groups (4.91, 3.03 and 1.99 g x d(-1), respectively) than for the 1.0- and 1.5-G groups. Body mass gain as a relative value did not differ between the 1.0-, 1.5-, and 2.0-G groups (1.5 +/- 0.2, 1.6 +/- 0.6 and 1.4 +/- 0.3 g x d(-1) x 100 g(-1) body mass, respectively), but was significantly less for the 2.5- and 3.5-G groups (1.1 +/- 0.6 and 0.8 +/- 0.3 g x d(-1) x 100 g(-1) body mass, respectively) than for the 1.0-, 1.5-, and 2.0-G groups. Absolute values and rate of body mass gain were reduced with increases in gravity. CONCLUSION: Exposure to hypergravity attenuates body mass gain in a dose-dependent manner, with a threshold possibly existing between 1.5- and 2.5-G for 10-wk-old male rats.

Morphometric changes in vagal nerves of fourth generation mice passage-bred in a 2-G environment.

INTRODUCTION: Previous studies have shown that microgravity induces both functional and structural adaptations in the autonomic nerves. Functional adaptation to hypergravity has also been reported, but structural change has not yet been isolated. The purpose of this study was to evaluate structural adaptation to hypergravity in the parasympathetic nerve. METHOD: We selected fourth generation mice which were passage-bred in a 2-G environment by cycles of coupling, delivery, and growth. Complete left cervical vagal nerves of these mice were studied in transverse sections by electron microscopy. The number of small (diameter < 5 microm, thin and light-stained myelin sheath) and large (diameter > 5 microm, thick and dark-stained myelin sheath) myelinated fibers was counted. RESULTS: The total number of all myelinated fibers (2 G: 795 +/- 103, 1 G: 644 +/- 60) and the number of small myelinated fibers (2 G: 657 +/- 95, 1 G: 522 +/- 66) were significantly greater in the 2-G mice than those in the 1-G mice (p < 0.05). The number of large myelinated fibers in the 2-G mice was greater than that in the 1-G mice, although it was not statistically significant (2 G: 138 +/- 15, 1-G: 122 +/- 16; p = 0.091). DISCUSSION: The results show that the autonomic nerves can adapt structurally to hypergravity. We contend that the present results are due to the fact that the mice were passage-bred. As far as we know, this is the first report to show an increase in myelinated fibers in autonomic nerves under prolonged exposure to an increased G environment.