The probability of double-strand breaks in giant DNA decreases markedly as the DNA concentration increases

DNA double-strand breaks (DSBs) represent a serious source of damage for all living things and thus there have been many quantitative studies of DSBs both in vivo and in vitro. Despite this fact, the processes that lead to their production have not yet been clearly understood, and there is no established theory that can account for the statistics of their production, in particular, the number of DSBs per base pair per unit Gy, here denoted by P1, which is the most important parameter for evaluating the degree of risk posed by DSBs. Here, using the single-molecule observation method with giant DNA molecules (166 kbp), we evaluate the number of DSBs caused by gamma-ray irradiation. We find that P1 is nearly inversely proportional to the DNA concentration above a certain threshold DNA concentration. A simple model that accounts for the marked decrease of P1 shows that it is necessary to consider the characteristics of giant DNA molecules as semiflexible polymers to interpret the intrinsic mechanism of DSBs

Subfoveal Choroidal Thickness and Axial Length in Preschool Children with Hyperopic Anisometropic Amblyopia

博士（医学）福島県立医科大

Topographic map reorganization in cat area 17 after early monocular retinal lesions

Neither discrete peripheral retinal lesions nor the normal optic disk produces obvious holes in one\u27s percept of the world because the visual brain appears to perceptually fill in these blind spots. Where in the visual brain or how this filling in occurs is not well understood. A prevailing hypothesis states that topographic map of visual cortex reorganizes after retinal lesions, which sews up the hole in the topographic map representing the deprived area of cortex (cortical scotoma) and may lead to perceptual filling in. Since the map reorganization does not typically occur unless retinotopically matched lesions are made in both eyes, we investigated the conditions in which monocular retinal lesions can induce comparable map reorganization. We found that following monocular retinal lesions, deprived neurons in cat area 17 can acquire new receptive fields if the lesion occurred relatively early in life (8 weeks of age) and the lesioned cats experienced a substantial period of recovery (\u3e3 years). Quantitative determination of the monocular and binocular response properties of reactivated units indicated that responses to the lesioned eye for such neurons were remarkably robust, and that the receptive-field properties for the two eyes were generally similar. Moreover, excitatory or inhibitory binocular interactions were found in the majority of experimental units when the two eyes were activated together. These results are consistent with the hypothesis that map reorganization after monocular retinal lesions require experience-dependent plasticity and may be involved in the perceptual filling in of blind spots due to retinal lesions early in life

Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.ArticleJOURNAL OF POULTRY SCIENCE. 51(1):87-95 (2014)journal articl

Binocular deficits associated with early alternating monocular defocus. II. Neurophysiological observations

Experiencing binocularly conflicting signals early in life dramatically alters the binocular responses of cortical neurons. Because visual cortex is highly plastic during a critical period of development, cortical deficits resulting from early abnormal visual experience often mirror the nature of interocular decorrelation of neural signals from the two eyes. In the preceding paper, we demonstrated that monkeys that experienced early alternating monocular defocus (-1.5, -3.0, or -6.0 D) show deficits in stereopsis that generally reflected the magnitude of imposed monocular defocus. Because these results indicated that alternating monocular defocus affected the higher spatial frequency components of visual scenes more severely, we employed microelectrode recording methods to investigate whether V1 neurons in these lens-reared monkeys exhibited spatial-frequency-dependent alterations in their binocular response properties. We found that a neuron\u27s sensitivity to interocular spatial phase disparity was reduced in the treated monkeys and that this reduction was generally more severe for units tuned to higher spatial frequencies. In the majority of the affected units, the disparity-sensitivity loss was associated with interocular differences in monocular receptive field properties. The present results suggest that the behavioral deficits in stereopsis produced by abnormal visual experience reflect at least in part the constraints imposed by alterations at the earliest stages of binocular cortical processing and support the hypothesis that the local disparity processing mechanisms in primates are spatially tuned and can be independently compromised by early abnormal visual experience

Adverse effect of donor-specific anti-human leukocyte antigen (HLA) antibodies directed at HLA-DP/-DQ on engraftment in cord blood transplantation

[Background aims] While donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) in the recipient before transplantation are associated with graft failure in cord-blood transplantation (CBT), effects of DSAs other than against HLA-A, -B or -DRB1 on transplantation outcomes remained poorly understood. [Methods] We retrospectively analyzed 567 single-unit CBT recipients to evaluate impact of DSAs against HLA-DP and -DQ on CBT outcomes. [Results] Among 143 recipients (25.2%) who had anti-HLA antibodies, nine harbored DSAs against HLA-DP or -DQ. DSAs against HLA-DP or -DQ were associated with a significantly lower neutrophil engraftment rate (55.6% versus 91.8%, P = 0.032) and with a marginally lower platelet engraftment rate (46.7% versus 75.3%, P = 0.128) at day 100 after transplantation, compared with patients without anti-HLA antibodies. Time to neutrophil and platelet engraftment in patients with DSAs for HLA-DP or -DQ was significantly longer than that in patients without anti-HLA antibodies (median, 25 versus 21 days, P = 0.002 in neutrophil; median 61 versus 46 days, P = 0.014 in platelet). Cumulative incidence of bacterial infection at day 100 was significantly greater (88.9% versus 57.1%, P = 0.024), and re-transplant-free survival was marginally lower (55.6% versus 76.8%, P = 0.132) in patients with DSAs against HLA-DP or -DQ, compared with those without anti-HLA antibodies. These findings suggest that DSAs against HLA-DP or -DQ lead to unfavorable engraftment, which may increase risk of bacterial infection, and reduce survival soon after CBT. [Conclusions] Our results suggest the importance of evaluating DSAs against HLA-DP and -DQ in recipients before selecting CB units

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

International audienceThe molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC beta-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The beta construct beta-AID dominant negative, which disrupts the RIM1-beta association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with beta in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs

Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS97xS99). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip–Syntaxin4 interaction