Adenylate Cyclase Mediates Olfactory Transduction of Amino Acid Responses in the Newt

It has been reported that amphibians can smell not only airborne odorants but also aminoacids. It is not clear, however, whether the signal transduction pathway of the amino acid responses is sameas that of volatile odorant responses. In this study, we use patch-clamp recordings of newt olfactory receptorneurons to show that amino acid (200 μM glutamic acid, acidic; 200 μM arginine, basic; 200 μM alanine orcysteine, neutral) responses are accompanied by inducing depolarizing currents. Moreover, responses toboth amino acids and forskolin, a stimulator of adenylate cyclase, were observed in the same cells, whichindicates that the cells responding to amino acids possess the cAMP-system. In addition, our EOG (electroolfactogram)studies show that forskolin attenuates not only responses to volatile odorants, but also those toamino acids. These data provide evidence that the cyclic AMP system might underlie the signal transductionpathway of amino acid responses in addition to volatile odorant responses

Efficient derivation and banking of clinical-grade human embryonic stem cell lines in accordance with Japanese regulations

[Introduction] We recently established clinical-grade human embryonic stem cell (hESC) line KthES11 in accordance with current good manufacturing practice standards in Japan. Despite this success, the establishment efficiency was very low at 7.1% in the first period. [Methods] To establish clinical-grade hESC lines, we used xeno-free chemically defined medium StemFit AK03N with the LM-E8 fragments instead of feeder cells. The protocol was then optimized, especially in the early culture phase. [Results] We established five hESC lines (KthES12, KthES13, KthES14, KthES15, and KthES16) with 45.5% efficiency. All five hESC lines showed typical hESC-like morphology, a normal karyotype, pluripotent state, and differentiation potential for all three germ layers. Furthermore, we developed efficient procedures to prepare master cell stocks for clinical-grade hESC lines and an efficient strategy for quality control testing. [Conclusions] Our master cell stocks of hESC lines may contribute to therapeutic applications using human pluripotent stem cells in Japan and other countries

Physiological Properties of Rod Photoreceptor Cells in Green-sensitive Cone Pigment Knock-in Mice

Rod and cone photoreceptor cells that are responsible for scotopic and photopic vision, respectively, exhibit photoresponses different from each other and contain similar phototransduction proteins with distinctive molecular properties. To investigate the contribution of the different molecular properties of visual pigments to the responses of the photoreceptor cells, we have generated knock-in mice in which rod visual pigment (rhodopsin) was replaced with mouse green-sensitive cone visual pigment (mouse green). The mouse green was successfully transported to the rod outer segments, though the expression of mouse green in homozygous retina was ∼11% of rhodopsin in wild-type retina. Single-cell recordings of wild-type and homozygous rods suggested that the flash sensitivity and the single-photon responses from mouse green were three to fourfold lower than those from rhodopsin after correction for the differences in cell volume and levels of several signal transduction proteins. Subsequent measurements using heterozygous rods expressing both mouse green and rhodopsin E122Q mutant, where these pigments in the same rod cells can be selectively irradiated due to their distinctive absorption maxima, clearly showed that the photoresponse of mouse green was threefold lower than that of rhodopsin. Noise analysis indicated that the rate of thermal activations of mouse green was 1.7 × 10−7 s−1, about 860-fold higher than that of rhodopsin. The increase in thermal activation of mouse green relative to that of rhodopsin results in only 4% reduction of rod photosensitivity for bright lights, but would instead be expected to severely affect the visual threshold under dim-light conditions. Therefore, the abilities of rhodopsin to generate a large single photon response and to retain high thermal stability in darkness are factors that have been necessary for the evolution of scotopic vision

Evaluation of Fatigue Properties under Four-point Bending and Fatigue Crack Propagation in Austenitic Stainless Steel with a Bimodal Harmonic Structure

Austenitic stainless steel (JIS-SUS304L) with a bimodal harmonic structure, which is defined as a coarse-grained structure surrounded by a network of fine grains, was fabricated using powder metallurgy to improve both the strength and ductility. Four-point bending fatigue tests and K-decreasing tests were conducted in air at room temperature under a stress ratio R of 0.1 to investigate fatigue crack propagation in SUS304L. The fatigue limit of this harmonic-structured material was higher than that of the material with a homogeneous coarse-grained structure. This is attributable to the formation of fine grains by mechanical milling and to the suppression of pore formation. In contrast, the threshold stress intensity range, DKth, for the harmonic-structured material was lower than that for the homogeneous coarse-grained material, while the crack growth rates, da/dN, were higher at comparable DK. These results can be attributed to a reduction in the effective threshold stress intensity range, DKeff,th, due to the presence of fine grains in the harmonic structure

p600 Plays Essential Roles in Fetal Development

p600 is a multifunctional protein implicated in cytoskeletal organization, integrin-mediated survival signaling, calcium-calmodulin signaling and the N-end rule pathway of ubiquitin-proteasome-mediated proteolysis. While push, the Drosophila counterpart of p600, is dispensable for development up to adult stage, the role of p600 has not been studied during mouse development. Here we generated p600 knockout mice to investigate the in vivo functions of p600. Interestingly, we found that homozygous deletion of p600 results in lethality between embryonic days 11.5 and 13.5 with severe defects in both embryo and placenta. Since p600 is required for placental development, we performed conditional disruption of p600, which deletes selectively p600 in the embryo but not in the placenta. The conditional mutant embryos survive longer than knockout embryos but ultimately die before embryonic day 14.5. The mutant embryos display severe cardiac problems characterized by ventricular septal defects and thin ventricular walls. These anomalies are associated with reduced activation of FAK and decreased expression of MEF2, which is regulated by FAK and plays a crucial role in cardiac development. Moreover, we observed pleiotropic defects in the liver and brain. In sum, our study sheds light on the essential roles of p600 in fetal development

Thirring Model as a Gauge Theory

We give another reformulation of the Thirring model (with four-fermion interaction of the current-current type) as a gauge theory and identify it with a gauge-fixed version of the corresponding gauge theory according to the Batalin-Fradkin formalism. Based on this formalism, we study the chiral symmetry breaking of the $D$-dimensional Thirring model ($2<D<4$) with $N$ flavors of 4-component fermions. By constructing the gauge covariant effective potential for the chiral order parameter, up to the leading order of $1/N$ expansion, we show the existence of the second order chiral phase transition and obtain explicitly the critical number of flavors $N_c$ (resp. critical four-fermion coupling $G_c$) as a function of the four-fermion coupling $G$ (resp. $N$), below (resp. above) which the chiral symmetry is spontaneously broken.Comment: 18 pages, LaTeX (revised final version, to be published in Nucl. Phys. B

Derivation of Human Differential Photoreceptor-like Cells from the Iris by Defined Combinations of CRX, RX and NEUROD

Examples of direct differentiation by defined transcription factors have been provided for beta-cells, cardiomyocytes and neurons. In the human visual system, there are four kinds of photoreceptors in the retina. Neural retina and iris-pigmented epithelium (IPE) share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons. We here define the transcription factor combinations that can determine human photoreceptor cell fate. Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Phototransduction-related genes as well as opsin genes were up-regulated in those cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that generated photoreceptor cells, induced by CRX, RX and NEUROD, responded to light. The response was an inward current instead of the typical outward current. These data suggest that photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX generate immature photoreceptors: and additional NEUROD promotes maturation. These findings contribute substantially to a major advance toward eventual cell-based therapy for retinal degenerative diseases

Testing the seismic quiescence hypothesis through retrospective trials of alarm-based earthquake prediction in the Kurile-Japan subduction zone

We make trial binary forecasts for the Kurile-Japan subduction zone for the period 1988-2014 by hypothesizing that seismic quiescence (i.e., the absence of earthquakes of M >= 5 for a minimum period of T-q) is a precursor of a large (7.5 = 9 years, within several tens of kilometers of the quiescence. We then attempt cross-validation, where we use half the data for training [i.e., optimization of (T-q, R, T-a)] and the remaining half for evaluation. With only four target earthquakes available for evaluation of the forecasts in each of the learning and evaluation periods, our forecast scheme did not pass the cross-validation test (with a criterion that the p-value is less than 5%). Hence, we cannot formally deny the possibility that our positive results for the overall period are a ghost arising from over-fitting. However, through detailed comparison of optimal models in the overall test with those in the cross-validation tests, we argue that severe over-fitting is unlikely involved for the modest G of similar to 2 obtained in the overall test. There is thus a reasonable chance that the presently tested type of quiescence will pass the cross-validation test when more target earthquakes become available in the near future. In the meantime, we find that G improves to similar to 5 when target earthquakes are limited to 8 <= M-w < 8.5, though we cannot say anything about the possible involvement of over-fitting because we have only three such very large target earthquakes