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Sample records for rod photoreceptor cgmp

  1. cGMP in Mouse Rods: the spatiotemporal dynamics underlying single photon responses

    Directory of Open Access Journals (Sweden)

    Owen P. Gross

    2015-03-01

    Full Text Available Vertebrate vision begins when retinal photoreceptors transduce photons into membrane hyperpolarization, which reduces glutamate release onto second-order neurons. In rod photoreceptors, transduction of single photons is achieved by a well-understood G-protein cascade that modulates cGMP levels, and in turn, cGMP-sensitive inward current. The spatial extent and depth of the decline in cGMP during the single photon response have been major issues in phototransduction research since the discovery that single photons elicit substantial and reproducible changes in membrane current. The spatial profile of cGMP decline during the single photon response affects signal gain, and thus may contribute to reduction of trial-to-trial fluctuations in the single photon response. Here we summarize the general principles of rod phototransduction, emphasizing recent advances in resolving the spatiotemporal dynamics of cGMP during the single photon response.

  2. The determination of total cGMP levels in rod outer segments from intact toad photoreceptors in response to light superimposed on background and to consecutive flashes: a second light flash accelerates the dark recovery rate of cGMP levels in control media, but not in Na(+)-free, low Ca2+ medium.

    Science.gov (United States)

    Cohen, A I; Blazynski, C

    1993-01-01

    In previous experiments we established that a light flash reduced cGMP levels of toad rod outer segments within the transduction time interval, but that recovery of the dark level of cGMP occurred more slowly than reported electrophysiological recovery of membrane potential. We now report that a second light flash accelerates the recovery rate of total cGMP following an initial flash, but that this acceleration is blocked in a medium which is both sodium and calcium deficient. We also noted that calcium deficiency only elevated cGMP levels when sodium was present. For other experiments, we recorded ERG or aspartate isolated PIII responses from eyecups or retinas mounted on our quick-freeze apparatus, the light stimuli originating from the double light-bench of the latter. Whereas background illumination depressed cGMP, no detectable further cGMP loss accompanied the electrical response to a flash superimposed on the background.

  3. Nrl is required for rod photoreceptor development.

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    Mears, A J; Kondo, M; Swain, P K; Takada, Y; Bush, R A; Saunders, T L; Sieving, P A; Swaroop, A

    2001-12-01

    The protein neural retina leucine zipper (Nrl) is a basic motif-leucine zipper transcription factor that is preferentially expressed in rod photoreceptors. It acts synergistically with Crx to regulate rhodopsin transcription. Missense mutations in human NRL have been associated with autosomal dominant retinitis pigmentosa. Here we report that deletion of Nrl in mice results in the complete loss of rod function and super-normal cone function, mediated by S cones. The photoreceptors in the Nrl-/- retina have cone-like nuclear morphology and short, sparse outer segments with abnormal disks. Analysis of retinal gene expression confirms the apparent functional transformation of rods into S cones in the Nrl-/- retina. On the basis of these findings, we postulate that Nrl acts as a 'molecular switch' during rod-cell development by directly modulating rod-specific genes while simultaneously inhibiting the S-cone pathway through the activation of Nr2e3.

  4. Control of ligand specificity in cyclic nucleotide-gated channels from rod photoreceptors and olfactory epithelium.

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    Altenhofen, W; Ludwig, J; Eismann, E; Kraus, W; Bönigk, W; Kaupp, U B

    1991-11-01

    Cyclic nucleotide-gated ionic channels in photoreceptors and olfactory sensory neurons are activated by binding of cGMP or cAMP to a receptor site on the channel polypeptide. By site-directed mutagenesis and functional expression of bovine wild-type and mutant channels in Xenopus oocytes, we have tested the hypothesis that an alanine/threonine difference in the cyclic nucleotide-binding site determines the specificity of ligand binding, as has been proposed for cyclic nucleotide-dependent protein kinases [Weber, I.T., Shabb, J.B. & Corbin, J.D. (1989) Biochemistry 28, 6122-6127]. The wild-type olfactory channel is approximately 25-fold more sensitive to both cAMP and cGMP than the wild-type rod photoreceptor channel, and both channels are 30- to 40-fold more sensitive to cGMP than to cAMP. Substitution of the respective threonine by alanine in the rod photoreceptor and olfactory channels decreases the cGMP sensitivity of channel activation 30-fold but little affects activation by cAMP. Substitution of threonine by serine, an amino acid that also carries a hydroxyl group, even improves cGMP sensitivity of the wild-type channels 2- to 5-fold. We conclude that the hydroxyl group of Thr-560 (rod) and Thr-537 (olfactory) forms an additional hydrogen bond with cGMP, but not cAMP, and thereby provides the structural basis for ligand discrimination in cyclic nucleotide-gated channels.

  5. Visual transduction in human rod photoreceptors.

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    Kraft, T W; Schneeweis, D M; Schnapf, J L

    1993-05-01

    1. Photocurrents were recorded with suction electrodes from rod photoreceptors of seven humans. 2. Brief flashes of light evoked transient outward currents of up to 20 pA. With increasing light intensity the peak response amplitude increased along an exponential saturation function. A half-saturating peak response was evoked by approximately sixty-five photoisomerizations. 3. Responses to brief dim flashes rose to a peak in about 200 ms. The waveform was roughly like the impulse response of a series of four to five low-pass filters. 4. The rising phases of the responses to flashes of increasing strength were found to fit with a biochemical model of phototransduction with an 'effective delay time' and 'characteristic time' of about 2 and 800 ms, respectively. 5. Spectral sensitivities were obtained over a wavelength range from 380 to 760 nm. The action spectrum, which peaked at 495 nm, followed the template described for photoreceptors in the macaque retina. Variation between rods in the position of the spectrum on the wavelength axis was small. 6. The scotopic luminosity function derived from human psychophysical experiments was found to agree well with the measured rod action spectrum after adjustments were made for lens absorption and photopigment self-screening in the intact eye. 7. Responses to steps of light rose monotonically to a maintained level, showing little or no relaxation. Nevertheless, the relationship between light intensity and steady-state response amplitude was shallower than that expected from simple response saturation. This is consistent with an adaptation mechanism acting on a rapid time scale. 8. Flash sensitivity fell with increasing intensities of background light according to Weber's law. Sensitivity was reduced twofold by lights evoking about 120 photoisomerizations per second. Background lights decreased the time to peak and the integration time of the flash response by up to 20%.

  6. Biophysical mechanism of transient retinal phototropism in rod photoreceptors

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    Zhao, Xiaohui; Thapa, Damber; Wang, Benquan; Gai, Shaoyan; Yao, Xincheng

    2016-03-01

    Oblique light stimulation evoked transient retinal phototropism (TRP) has been recently detected in frog and mouse retinas. High resolution microscopy of freshly isolated retinas indicated that the TRP is predominated by rod photoreceptors. Comparative confocal microscopy and optical coherence tomography (OCT) revealed that the TRP predominantly occurred from the photoreceptor outer segment (OS). However, biophysical mechanism of rod OS change is still unknown. In this study, frog retinal slices, which open a cross section of retinal photoreceptor and other functional layers, were used to test the effect of light stimulation on rod OS. Near infrared light microscopy was employed to monitor photoreceptor changes in retinal slices stimulated by a rectangular-shaped visible light flash. Rapid rod OS length change was observed after the stimulation delivery. The magnitude and direction of the rod OS change varied with the position of the rods within the stimulated area. In the center of stimulated region the length of the rod OS shrunk, while in the peripheral region the rod OS tip swung towards center region in the plane perpendicular to the incident stimulus light. Our experimental result and theoretical analysis suggest that the observed TRP may reflect unbalanced disc-shape change due to localized pigment bleaching. Further investigation is required to understand biochemical mechanism of the observed rod OS kinetics. Better study of the TRP may provide a noninvasive biomarker to enable early detection of age-related macular degeneration (AMD) and other diseases that are known to produce retinal photoreceptor dysfunctions.

  7. Stimulus-evoked outer segment changes in rod photoreceptors

    Science.gov (United States)

    Zhao, Xiaohui; Thapa, Damber; Wang, Benquan; Lu, Yiming; Gai, Shaoyan; Yao, Xincheng

    2016-06-01

    Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation.

  8. Adaptive potentiation in rod photoreceptors after light exposure

    OpenAIRE

    McKeown, Alex S; Kraft, Timothy W.

    2014-01-01

    Photoreceptors adapt to changes in illumination by altering transduction kinetics and sensitivity, thereby extending their working range. We describe a previously unknown form of rod photoreceptor adaptation in wild-type (WT) mice that manifests as a potentiation of the light response after periods of conditioning light exposure. We characterize the stimulus conditions that evoke this graded hypersensitivity and examine the molecular mechanisms of adaptation underlying the phenomenon. After e...

  9. NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors.

    Science.gov (United States)

    Kim, Jung-Woong; Yang, Hyun-Jin; Brooks, Matthew John; Zelinger, Lina; Karakülah, Gökhan; Gotoh, Norimoto; Boleda, Alexis; Gieser, Linn; Giuste, Felipe; Whitaker, Dustin Thad; Walton, Ashley; Villasmil, Rafael; Barb, Jennifer Joanna; Munson, Peter Jonathan; Kaya, Koray Dogan; Chaitankar, Vijender; Cogliati, Tiziana; Swaroop, Anand

    2016-11-22

    Gene regulatory networks (GRNs) guiding differentiation of cell types and cell assemblies in the nervous system are poorly understood because of inherent complexities and interdependence of signaling pathways. Here, we report transcriptome dynamics of differentiating rod photoreceptors in the mammalian retina. Given that the transcription factor NRL determines rod cell fate, we performed expression profiling of developing NRL-positive (rods) and NRL-negative (S-cone-like) mouse photoreceptors. We identified a large-scale, sharp transition in the transcriptome landscape between postnatal days 6 and 10 concordant with rod morphogenesis. Rod-specific temporal DNA methylation corroborated gene expression patterns. De novo assembly and alternative splicing analyses revealed previously unannotated rod-enriched transcripts and the role of NRL in transcript maturation. Furthermore, we defined the relationship of NRL with other transcriptional regulators and downstream cognate effectors. Our studies provide the framework for comprehensive system-level analysis of the GRN underlying the development of a single sensory neuron, the rod photoreceptor. Published by Elsevier Inc.

  10. Adaptive potentiation in rod photoreceptors after light exposure.

    Science.gov (United States)

    McKeown, Alex S; Kraft, Timothy W

    2014-06-01

    Photoreceptors adapt to changes in illumination by altering transduction kinetics and sensitivity, thereby extending their working range. We describe a previously unknown form of rod photoreceptor adaptation in wild-type (WT) mice that manifests as a potentiation of the light response after periods of conditioning light exposure. We characterize the stimulus conditions that evoke this graded hypersensitivity and examine the molecular mechanisms of adaptation underlying the phenomenon. After exposure to periods of saturating illumination, rods show a 10-35% increase in circulating dark current, an adaptive potentiation (AP) to light exposure. This potentiation grows as exposure to light is extended up to 3 min and decreases with longer exposures. Cells return to their initial dark-adapted sensitivity with a time constant of recovery of ∼7 s. Halving the extracellular Mg concentration prolongs the adaptation, increasing the time constant of recovery to 13.3 s, but does not affect the magnitude of potentiation. In rods lacking guanylate cyclase activating proteins 1 and 2 (GCAP(-/-)), AP is more than doubled compared with WT rods, and halving the extracellular Mg concentration does not affect the recovery time constant. Rods from a mouse expressing cyclic nucleotide-gated channels incapable of binding calmodulin also showed a marked increase in the amplitude of AP. Application of an insulin-like growth factor-1 receptor (IGF-1R) kinase inhibitor (Tyrphostin AG1024) blocked AP, whereas application of an insulin receptor kinase inhibitor (HNMPA(AM)3) failed to do so. A broad-acting tyrosine phosphatase inhibitor (orthovanadate) also blocked AP. Our findings identify a unique form of adaptation in photoreceptors, so that they show transient hypersensitivity to light, and are consistent with a model in which light history, acting via the IGF-1R, can increase the sensitivity of rod photoreceptors, whereas the photocurrent overshoot is regulated by Ca-calmodulin and Ca(2

  11. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness.

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    Wiley, Luke A; Burnight, Erin R; DeLuca, Adam P; Anfinson, Kristin R; Cranston, Cathryn M; Kaalberg, Emily E; Penticoff, Jessica A; Affatigato, Louisa M; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-07-29

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

  12. Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse.

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    Arshavsky, V Y; Dumke, C L; Bownds, M D

    1992-12-05

    The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range

  13. Deafferented Adult Rod Bipolar Cells Create New Synapses with Photoreceptors to Restore Vision.

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    Beier, Corinne; Hovhannisyan, Anahit; Weiser, Sydney; Kung, Jennifer; Lee, Seungjun; Lee, Dae Yeong; Huie, Philip; Dalal, Roopa; Palanker, Daniel; Sher, Alexander

    2017-04-26

    Upon degeneration of photoreceptors in the adult retina, interneurons, including bipolar cells, exhibit a plastic response leading to their aberrant rewiring. Photoreceptor reintroduction has been suggested as a potential approach to sight restoration, but the ability of deafferented bipolar cells to establish functional synapses with photoreceptors is poorly understood. Here we use photocoagulation to selectively destroy photoreceptors in adult rabbits while preserving the inner retina. We find that rods and cones shift into the ablation zone over several weeks, reducing the blind spot at scotopic and photopic luminances. During recovery, rod and cone bipolar cells exhibit markedly different responses to deafferentation. Rod bipolar cells extend their dendrites to form new synapses with healthy photoreceptors outside the lesion, thereby restoring visual function in the deafferented retina. Secretagogin-positive cone bipolar cells did not exhibit such obvious dendritic restructuring. These findings are encouraging to the idea of photoreceptor reintroduction for vision restoration in patients blinded by retinal degeneration. At the same time, they draw attention to the postsynaptic side of photoreceptor reintroduction; various bipolar cell types, representing different visual pathways, vary in their response to the photoreceptor loss and in their consequent dendritic restructuring.SIGNIFICANCE STATEMENT Loss of photoreceptors during retinal degeneration results in permanent visual impairment. Strategies for vision restoration based on the reintroduction of photoreceptors inherently rely on the ability of the remaining retinal neurons to correctly synapse with new photoreceptors. We show that deafferented bipolar cells in the adult mammalian retina can reconnect to rods and cones and restore retinal sensitivity at scotopic and photopic luminances. Rod bipolar cells extend their dendrites to form new synapses with healthy rod photoreceptors. These findings support the

  14. Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.

    Directory of Open Access Journals (Sweden)

    Natalia López-del Hoyo

    Full Text Available Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout and gain-of-function (transgenic overexpression mouse models of GCAP2. Rod synaptic ribbons in GCAPs-/- mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs-/- background (GCAP2 expression in the absence of endogenous GCAP1 had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of

  15. Multiple rod-cone and cone-rod photoreceptor transmutations in snakes: evidence from visual opsin gene expression.

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    Simões, Bruno F; Sampaio, Filipa L; Loew, Ellis R; Sanders, Kate L; Fisher, Robert N; Hart, Nathan S; Hunt, David M; Partridge, Julian C; Gower, David J

    2016-01-27

    In 1934, Gordon Walls forwarded his radical theory of retinal photoreceptor 'transmutation'. This proposed that rods and cones used for scotopic and photopic vision, respectively, were not fixed but could evolve into each other via a series of morphologically distinguishable intermediates. Walls' prime evidence came from series of diurnal and nocturnal geckos and snakes that appeared to have pure-cone or pure-rod retinas (in forms that Walls believed evolved from ancestors with the reverse complement) or which possessed intermediate photoreceptor cells. Walls was limited in testing his theory because the precise identity of visual pigments present in photoreceptors was then unknown. Subsequent molecular research has hitherto neglected this topic but presents new opportunities. We identify three visual opsin genes, rh1, sws1 and lws, in retinal mRNA of an ecologically and taxonomically diverse sample of snakes central to Walls' theory. We conclude that photoreceptors with superficially rod- or cone-like morphology are not limited to containing scotopic or photopic opsins, respectively. Walls' theory is essentially correct, and more research is needed to identify the patterns, processes and functional implications of transmutation. Future research will help to clarify the fundamental properties and physiology of photoreceptors adapted to function in different light levels.

  16. Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL.

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    Oh, Edwin C T; Khan, Naheed; Novelli, Elena; Khanna, Hemant; Strettoi, Enrica; Swaroop, Anand

    2007-01-30

    Networks of transcriptional regulatory proteins dictate specification of neural lineages from multipotent retinal progenitors. Rod photoreceptor differentiation requires the basic motif-leucine zipper (bZIP) transcription factor NRL, because loss of Nrl in mice (Nrl-/-) results in complete transformation of rods to functional cones. To examine the role of NRL in cell fate determination, we generated transgenic mice that express Nrl under the control of Crx promoter in postmitotic photoreceptor precursors of WT and Nrl-/- retina. We show that NRL expression, in both genetic backgrounds, leads to a functional retina with only rod photoreceptors. The absence of cones does not alter retinal lamination, although cone synaptic circuitry is now recruited by rods. Ectopic expression of NRL in developing cones can also induce rod-like characteristics and partially suppress cone-specific gene expression. We show that NRL is associated with specific promoter sequences in Thrb (encoding TRbeta2 transcription factor required for M-cone differentiation) and S-opsin and may, therefore, directly participate in transcriptional suppression of cone development. Our studies establish that NRL is not only essential but is sufficient for rod differentiation and that postmitotic photoreceptor precursors are competent to make binary decisions during early retinogenesis.

  17. Generation of a genetically encoded marker of rod photoreceptor outer segment growth and renewal

    Directory of Open Access Journals (Sweden)

    John J. Willoughby

    2011-10-01

    Vertebrate photoreceptors are specialized light sensing neurons. The photoreceptor outer segment is a highly modified cilium where photons of light are transduced into a chemical and electrical signal. The outer segment has the typical cilary axoneme but, in addition, it has a large number of densely packed, stacked, intramembranous discs. The molecular and cellular mechanisms that contribute to vertebrate photoreceptor outer segment morphogenesis are still largely unknown. Unlike typical cilia, the outer segment is continuously regenerated or renewed throughout the life of the animal through the combined process of distal outer segment shedding and proximal outer segment growth. The process of outer segment renewal was discovered over forty years ago, but we still lack an understanding of how photoreceptors renew their outer segments and few, if any, molecular mechanisms that regulate outer segment growth or shedding have been described. Our lack of progress in understanding how photoreceptors renew their outer segments has been hampered by the difficulty in measuring rates of renewal. We have created a new method that uses heat-shock induction of a fluorescent protein that can be used to rapidly measure outer segment growth rates. We describe this method, the stable transgenic line we created, and the growth rates observed in larval and adult rod photoreceptors using this new method. This new method will allow us to begin to define the genetic and molecular mechanisms that regulate rod outer segment renewal, a crucial aspect of photoreceptor function and, possibly, viability.

  18. miR Cluster 143/145 Directly Targets Nrl and Regulates Rod Photoreceptor Development.

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    Sreekanth, Sreekumaran; Rasheed, Vazhanthodi A; Soundararajan, Lalitha; Antony, Jayesh; Saikia, Minakshi; Sivakumar, Krishnankutty Chandrika; Das, Ani V

    2016-11-23

    Retinal histogenesis requires coordinated and temporal functioning of factors by which different cell types are generated from multipotent progenitors. Development of rod photoreceptors is regulated by multiple transcription factors, and Nrl is one of the major factors involved in their fate specification. Presence or absence of Nrl at the postnatal stages decides the generation of cone photoreceptors or other later retinal cells. This suggests the need for regulated expression of Nrl in order to accelerate the generation of other cell types during retinal development. We found that miR cluster 143/145, comprising miR-143 and miR-145, targets and imparts a posttranscriptional inhibition of Nrl. Expression of both miRNAs was differentially regulated during retinal development and showed least expression at PN1 stage in which most of the rod photoreceptors are generated. Downregulation of rod photoreceptor regulators and markers upon miR cluster 143/145 overexpression demonstrated that this cluster indeed negatively regulates rod photoreceptors. Further, we prove that Nrl positively regulates miR cluster 143/145, thus establishing a feedback loop regulatory mechanism. This may be one possible mechanism by which Nrl is posttranscriptionally regulated to facilitate the generation of other cell types in retina.

  19. Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis.

    Directory of Open Access Journals (Sweden)

    Hong Hao

    Full Text Available A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.

  20. Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis.

    Science.gov (United States)

    Hao, Hong; Kim, Douglas S; Klocke, Bernward; Johnson, Kory R; Cui, Kairong; Gotoh, Norimoto; Zang, Chongzhi; Gregorski, Janina; Gieser, Linn; Peng, Weiqun; Fann, Yang; Seifert, Martin; Zhao, Keji; Swaroop, Anand

    2012-01-01

    A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.

  1. Transcriptional regulation of rod photoreceptor homeostasis revealed by in vivo NRL targetome analysis.

    Directory of Open Access Journals (Sweden)

    Hong Hao

    Full Text Available A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.

  2. Irradiance encoding in the suprachiasmatic nuclei by rod and cone photoreceptors.

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    van Diepen, Hester C; Ramkisoensing, Ashna; Peirson, Stuart N; Foster, Russell G; Meijer, Johanna H

    2013-10-01

    Light information is transmitted to the central clock of the suprachiasmatic nuclei (SCN) for daily synchronization to the external solar cycle. Essential for synchronization is the capacity of SCN neurons to respond in a sustained and irradiance-dependent manner to light. Melanopsin has been considered to mediate this photosensory task of irradiance detection. By contrast, the contribution of the classical photoreceptors in irradiance encoding is less clear. Here we investigate the role of classical photoreceptors by in vivo electrophysiological responses in freely moving animals to specific wavelengths of light (UV, λmax 365 nm; blue, λmax 467 nm; and green, λmax 505 nm) in both melanopsin-deficient (Opn4(-/-)) mice and mice lacking rods and cones (rd/rd cl). Short- and long-wavelength light induced sustained irradiance-dependent responses in congenic wild-type mice (+19.6%). Unexpectedly, sustained responses to light persisted in Opn4(-/-) mice (+18.4%). These results provide unambiguous evidence that classical photoreceptors can transmit irradiance information to the SCN. In addition, at light intensities that would stimulate rod and cone photoreceptors, the SCN of rd/rd cl mice showed greatly reduced sustained responses to light (+7.8%). Collectively, our data demonstrate a role for classical photoreceptors in illuminance detection by the SCN.

  3. Regulation of Noncoding Transcriptome in Developing Photoreceptors by Rod Differentiation Factor NRL.

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    Zelinger, Lina; Karakülah, Gökhan; Chaitankar, Vijender; Kim, Jung-Woong; Yang, Hyun-Jin; Brooks, Matthew J; Swaroop, Anand

    2017-09-01

    Transcriptome analysis by next generation sequencing allows qualitative and quantitative profiling of expression patterns associated with development and disease. However, most transcribed sequences do not encode proteins, and little is known about the functional relevance of noncoding (nc) transcriptome in neuronal subtypes. The goal of this study was to perform a comprehensive analysis of long noncoding (lncRNAs) and antisense (asRNAs) RNAs expressed in mouse retinal photoreceptors. Transcriptomic profiles were generated at six developmental time points from flow-sorted Nrlp-GFP (rods) and Nrlp-GFP;Nrl-/- (S-cone like) mouse photoreceptors. Bioinformatic analysis was performed to identify novel noncoding transcripts and assess their regulation by rod differentiation factor neural retina leucine zipper (NRL). In situ hybridization (ISH) was used for validation and cellular localization. NcRNA profiles demonstrated dynamic yet specific expression signature and coexpression clusters during rod development. In addition to currently annotated 586 lncRNAs and 454 asRNAs, we identified 1037 lncRNAs and 243 asRNAs by de novo assembly. Of these, 119 lncRNAs showed altered expression in the absence of NRL and included NRL binding sites in their promoter/enhancer regions. ISH studies validated the expression of 24 lncRNAs (including 12 previously unannotated) and 4 asRNAs in photoreceptors. Coexpression analysis demonstrated 63 functional modules and 209 significant antisense-gene correlations, allowing us to predict possible role of these lncRNAs in rods. Our studies reveal coregulation of coding and noncoding transcripts in rod photoreceptors by NRL and establish the framework for deciphering the function of ncRNAs during retinal development.

  4. Rod photoreceptors express GPR55 in the adult vervet monkey retina

    DEFF Research Database (Denmark)

    Bouskila, Joseph; Javadi, Pasha; Casanova, Christian

    2013-01-01

    Cannabinoids exert their actions mainly through two receptors, the cannabinoid CB1 receptor (CB1R) and cannabinoid CB2 receptor (CB2R). In recent years, the G-protein coupled receptor 55 (GPR55) was suggested as a cannabinoid receptor based on its activation by anandamide and tetrahydrocannabinol...... components (Müller cells). The aim of this study was to determine the expression pattern of GPR55 in the monkey retina by using confocal microscopy. Our results show that GPR55 is strictly localized in the photoreceptor layer of the extrafoveal portion of the retina. Co-immunolabeling of GPR55 with rhodopsin......, the photosensitive pigment in rods, revealed a clear overlap of expression throughout the rod structure with most prominent staining in the inner segments. Additionally, double-label of GPR55 with calbindin, a specific marker for cone photoreceptors in the primate retina, allowed us to exclude expression of GPR55...

  5. Rod photoreceptors express GPR55 in the adult vervet monkey retina.

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    Joseph Bouskila

    Full Text Available Cannabinoids exert their actions mainly through two receptors, the cannabinoid CB1 receptor (CB1R and cannabinoid CB2 receptor (CB2R. In recent years, the G-protein coupled receptor 55 (GPR55 was suggested as a cannabinoid receptor based on its activation by anandamide and tetrahydrocannabinol. Yet, its formal classification is still a matter of debate. CB1R and CB2R expression patterns are well described for rodent and monkey retinas. In the monkey retina, CB1R has been localized in its neural (cone photoreceptor, horizontal, bipolar, amacrine and ganglion cells and CB2R in glial components (Müller cells. The aim of this study was to determine the expression pattern of GPR55 in the monkey retina by using confocal microscopy. Our results show that GPR55 is strictly localized in the photoreceptor layer of the extrafoveal portion of the retina. Co-immunolabeling of GPR55 with rhodopsin, the photosensitive pigment in rods, revealed a clear overlap of expression throughout the rod structure with most prominent staining in the inner segments. Additionally, double-label of GPR55 with calbindin, a specific marker for cone photoreceptors in the primate retina, allowed us to exclude expression of GPR55 in cones. The labeling of GPR55 in rods was further assessed with a 3D visualization in the XZ and YZ planes thus confirming its exclusive expression in rods. These results provide data on the distribution of GPR55 in the monkey retina, different than CB1R and CB2R. The presence of GPR55 in rods suggests a function of this receptor in scotopic vision that needs to be demonstrated.

  6. Low-conductance HCN1 ion channels augment the frequency response of rod and cone photoreceptors.

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    Barrow, Andrew J; Wu, Samuel M

    2009-05-06

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are expressed in several tissues throughout the body, including the heart, the CNS, and the retina. HCN channels are found in many neurons in the retina, but their most established role is in generating the hyperpolarization-activated current, I(h), in photoreceptors. This current makes the light response of rod and cone photoreceptors more transient, an effect similar to that of a high-pass filter. A unique property of HCN channels is their small single-channel current, which is below the thermal noise threshold of measuring electronics. We use nonstationary fluctuation analysis (NSFA) in the intact retina to estimate the conductance of single HCN channels, revealing a conductance of approximately 650 fS in both rod and cone photoreceptors. We also analyze the properties of HCN channels in salamander rods and cones, from the biophysical to the functional level, showing that HCN1 is the predominant isoform in both cells, and demonstrate how HCN1 channels speed up the light response of both rods and cones under distinct adaptational conditions. We show that in rods and cones, HCN channels increase the natural frequency response of single cells by modifying the photocurrent input, which is limited in its frequency response by the speed of a molecular signaling cascade. In doing so, HCN channels form the first of several systems in the retina that augment the speed of the visual response, allowing an animal to perceive visual stimuli that change more quickly than the underlying photocurrent.

  7. Notch and Wnt signaling mediated rod photoreceptor regeneration by Muller cells in adult mammalian retina.

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    Carolina Beltrame Del Debbio

    Full Text Available BACKGROUND: Evidence emerging from a variety of approaches used in different species suggests that Müller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched Müller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that Notch and Wnt signaling activate Müller cells through their canonical pathways and that a rare subset of activated Müller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated Müller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate Müller cells. The number of activated Müller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception. CONCLUSION/SIGNIFICANCE: In summary, our results provide a proof of principle for non-neurotoxin-mediated activation of Müller cells through Notch and Wnt signaling toward the regeneration of rod photoreceptors.

  8. Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon

    OpenAIRE

    Natalia López-del Hoyo; Lucrezia Fazioli; Santiago López-Begines; Laura Fernández-Sánchez; Nicolás Cuenca; Jordi Llorens; Pedro de la Villa; Ana Méndez

    2012-01-01

    Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based o...

  9. The giant mottled eel, Anguilla marmorata, uses blue-shifted rod photoreceptors during upstream migration.

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    Feng-Yu Wang

    Full Text Available Catadromous fishes migrate between ocean and freshwater during particular phases of their life cycle. The dramatic environmental changes shape their physiological features, e.g. visual sensitivity, olfactory ability, and salinity tolerance. Anguilla marmorata, a catadromous eel, migrates upstream on dark nights, following the lunar cycle. Such behavior may be correlated with ontogenetic changes in sensory systems. Therefore, this study was designed to identify changes in spectral sensitivity and opsin gene expression of A. marmorata during upstream migration. Microspectrophotometry analysis revealed that the tropical eel possesses a duplex retina with rod and cone photoreceptors. The λmax of rod cells are 493, 489, and 489 nm in glass, yellow, and wild eels, while those of cone cells are 508, and 517 nm in yellow, and wild eels, respectively. Unlike European and American eels, Asian eels exhibited a blue-shifted pattern of rod photoreceptors during upstream migration. Quantitative gene expression analyses of four cloned opsin genes (Rh1f, Rh1d, Rh2, and SWS2 revealed that Rh1f expression is dominant at all three stages, while Rh1d is expressed only in older yellow eel. Furthermore, sequence comparison and protein modeling studies implied that a blue shift in Rh1d opsin may be induced by two known (N83, S292 and four putative (S124, V189, V286, I290 tuning sites adjacent to the retinal binding sites. Finally, expression of blue-shifted Rh1d opsin resulted in a spectral shift in rod photoreceptors. Our observations indicate that the giant mottled eel is color-blind, and its blue-shifted scotopic vision may influence its upstream migration behavior and habitat choice.

  10. Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles

    Energy Technology Data Exchange (ETDEWEB)

    Asteriti, Sabrina [Dept. of Translational Research, University of Pisa, Pisa (Italy); Dal Cortivo, Giuditta [Dept. of Life Sciences and Reproduction, University of Verona, Strada Le Grazie 8, Verona (Italy); Pontelli, Valeria [Dept. of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, Verona (Italy); Cangiano, Lorenzo [Dept. of Translational Research, University of Pisa, Pisa (Italy); Buffelli, Mario, E-mail: mario.buffelli@univr.it [Dept. of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, Verona (Italy); Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona (Italy); Dell’Orco, Daniele, E-mail: daniele.dellorco@univr.it [Dept. of Life Sciences and Reproduction, University of Verona, Strada Le Grazie 8, Verona (Italy); Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona (Italy)

    2015-06-12

    The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer. - Highlights: • Recombinant proteins encapsulated in nano-sized liposomes injected intravitreally reach retinal photoreceptors. • The phototransduction cascade in rods is modulated by the liposome content. • Mathematical modeling predicts the alteration of the photoresponses following liposome fusion.

  11. Recruitment of Rod Photoreceptors from Short-Wavelength-Sensitive Cones during the Evolution of Nocturnal Vision in Mammals.

    Science.gov (United States)

    Kim, Jung-Woong; Yang, Hyun-Jin; Oel, Adam Phillip; Brooks, Matthew John; Jia, Li; Plachetzki, David Charles; Li, Wei; Allison, William Ted; Swaroop, Anand

    2016-06-20

    Vertebrate ancestors had only cone-like photoreceptors. The duplex retina evolved in jawless vertebrates with the advent of highly photosensitive rod-like photoreceptors. Despite cones being the arbiters of high-resolution color vision, rods emerged as the dominant photoreceptor in mammals during a nocturnal phase early in their evolution. We investigated the evolutionary and developmental origins of rods in two divergent vertebrate retinas. In mice, we discovered genetic and epigenetic vestiges of short-wavelength cones in developing rods, and cell-lineage tracing validated the genesis of rods from S cones. Curiously, rods did not derive from S cones in zebrafish. Our study illuminates several questions regarding the evolution of duplex retina and supports the hypothesis that, in mammals, the S-cone lineage was recruited via the Maf-family transcription factor NRL to augment rod photoreceptors. We propose that this developmental mechanism allowed the adaptive exploitation of scotopic niches during the nocturnal bottleneck early in mammalian evolution. Published by Elsevier Inc.

  12. Mechanisms, pools, and sites of spontaneous vesicle release at synapses of rod and cone photoreceptors.

    Science.gov (United States)

    Cork, Karlene M; Van Hook, Matthew J; Thoreson, Wallace B

    2016-08-01

    Photoreceptors have depolarized resting potentials that stimulate calcium-dependent release continuously from a large vesicle pool but neurons can also release vesicles without stimulation. We characterized the Ca(2+) dependence, vesicle pools, and release sites involved in spontaneous release at photoreceptor ribbon synapses. In whole-cell recordings from light-adapted horizontal cells (HCs) of tiger salamander retina, we detected miniature excitatory post-synaptic currents (mEPSCs) when no stimulation was applied to promote exocytosis. Blocking Ca(2+) influx by lowering extracellular Ca(2+) , by application of Cd(2+) and other agents reduced the frequency of mEPSCs but did not eliminate them, indicating that mEPSCs can occur independently of Ca(2+) . We also measured release presynaptically from rods and cones by examining quantal glutamate transporter anion currents. Presynaptic quantal event frequency was reduced by Cd(2+) or by increased intracellular Ca(2+) buffering in rods, but not in cones, that were voltage clamped at -70 mV. By inhibiting the vesicle cycle with bafilomycin, we found the frequency of mEPSCs declined more rapidly than the amplitude of evoked excitatory post-synaptic currents (EPSCs) suggesting a possible separation between vesicle pools in evoked and spontaneous exocytosis. We mapped sites of Ca(2+) -independent release using total internal reflectance fluorescence (TIRF) microscopy to visualize fusion of individual vesicles loaded with dextran-conjugated pHrodo. Spontaneous release in rods occurred more frequently at non-ribbon sites than evoked release events. The function of Ca(2+) -independent spontaneous release at continuously active photoreceptor synapses remains unclear, but the low frequency of spontaneous quanta limits their impact on noise.

  13. Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina.

    Science.gov (United States)

    Zhou, Liang; Wang, Wei; Liu, Yongqing; Fernandez de Castro, Juan; Ezashi, Toshihiko; Telugu, Bhanu Prakash V L; Roberts, R Michael; Kaplan, Henry J; Dean, Douglas C

    2011-06-01

    Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.

  14. The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton.

    Science.gov (United States)

    Reidel, Boris; Goldmann, Tobias; Giessl, Andreas; Wolfrum, Uwe

    2008-10-01

    In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subsequently analyzed by light and electron microscopy. The application of cytoskeletal drugs differentially affected the cytoskeleton in photoreceptor compartments. During dark adaptation the depolymerization of microtubules as well as actin filaments disrupted the translocation of arrestin and transducin in rod photoreceptor cells. During light adaptation only the delivery of arrestin within the outer segment was impaired after destabilization of microtubules. Movements of transducin and arrestin required intact cytoskeletal elements in dark adapting cells. However, diffusion might be sufficient for the fast molecular movements observed as cells adapt to light. These findings indicate that different molecular translocation mechanisms are responsible for the dark and light associated translocations of arrestin and transducin in rod photoreceptor cells.

  15. Niflumic acid reduces the hyperpolarization-activated current (I(h)) in rod photoreceptor cells.

    Science.gov (United States)

    Satoh, T O; Yamada, M

    2001-08-01

    We examined the effects of niflumic acid (NFA), a chloride channel blocker, on the hyperpolarization-activated current (I(h)) in newt rod photoreceptors. At 100 microM, NFA delayed the activation of I(h) induced by hyperpolarizing voltage pulses to -83 mV from a holding potential of -43 mV, and reduced the steady-state current. However, reduction by NFA was weakened when I(h) was activated by hyperpolarizing steps to -123 mV, suggesting that these effects were voltage-dependent. The suppressive effects of NFA on I(h) were accompanied by a negative shift in activation voltage. NFA also delayed the relaxation of I(h) tail currents, showing that this drug also inhibited deactivation of the current. The reversal potential and the fully activated conductance were not affected. These observations suggest that NFA reduces I(h) by modifying the gating kinetics of the underlying channels. The suppressive actions of NFA remained when intracellular Ca2+ was strongly chelated, and the failure of suppression by NFA in inside-out patches suggests that the agent may act on the I(h) channel from the extracellular side. These results, obtained in rod photoreceptors, are consistent with similar effects of NFA on I(f) in cardiac myocytes, suggesting that both currents share similar pharmacological properties.

  16. Patterns of cell proliferation and rod photoreceptor differentiation in shark retinas.

    Science.gov (United States)

    Ferreiro-Galve, Susana; Rodríguez-Moldes, Isabel; Anadón, Ramón; Candal, Eva

    2010-01-01

    We studied the pattern of cell proliferation and its relation with photoreceptor differentiation in the embryonic and postembryonic retina of two elasmobranchs, the lesser spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus). Cell proliferation was studied with antibodies raised against proliferating cell nuclear antigen (PCNA) and phospho-histone-H3, and early photoreceptor differentiation with an antibody raised against rod opsin. As regards the spatiotemporal distribution of PCNA-immunoreactive cells, our results reveal a gradual loss of PCNA that coincides in a spatiotemporal sequence with the gradient of layer maturation. The presence of a peripheral growth zone containing pure-proliferating retinal progenitors (the ciliary marginal zone) in the adult retina matches with the general pattern observed in other groups of gnathostomous fishes. However, in the shark retina the generation of new cells is not restricted to the ciliary marginal zone but also occurs in retinal areas that contain differentiated cells: (1) in a transition zone that lies between the pure-proliferating ciliary marginal zone and the central (layered) retina; (2) in the differentiating central area up to prehatching embryos where large amounts of PCNA-positive cells were observed even in the inner and outer nuclear layers; (3) and in the retinal pigment epithelium of prehatching embryos. Rod opsin immunoreactivity was observed in both species when the outer plexiform layer begins to be recognized in the central retina and, as we previously observed in trout, coincided temporally with the weakening in PCNA labelling.

  17. Fasudil, a Clinically Used ROCK Inhibitor, Stabilizes Rod Photoreceptor Synapses after Retinal Detachment.

    Science.gov (United States)

    Townes-Anderson, Ellen; Wang, Jianfeng; Halász, Éva; Sugino, Ilene; Pitler, Amy; Whitehead, Ian; Zarbin, Marco

    2017-06-01

    Retinal detachment disrupts the rod-bipolar synapse in the outer plexiform layer by retraction of rod axons. We showed that breakage is due to RhoA activation whereas inhibition of Rho kinase (ROCK), using Y27632, reduces synaptic damage. We test whether the ROCK inhibitor fasudil, used for other clinical applications, can prevent synaptic injury after detachment. Detachments were made in pigs by subretinal injection of balanced salt solution (BSS) or fasudil (1, 10 mM). In some animals, fasudil was injected intravitreally after BSS-induced detachment. After 2 to 4 hours, retinae were fixed for immunocytochemistry and confocal microscopy. Axon retraction was quantified by imaging synaptic vesicle label in the outer nuclear layer. Apoptosis was analyzed using propidium iodide staining. For biochemical analysis by Western blotting, retinal explants, detached from retinal pigmented epithelium, were cultured for 2 hours. Subretinal injection of fasudil (10 mM) reduced retraction of rod spherules by 51.3% compared to control detachments (n = 3 pigs, P = 0.002). Intravitreal injection of 10 mM fasudil, a more clinically feasible route of administration, also reduced retraction (28.7%, n = 5, P ROCK, was decreased with 30 μM fasudil (n = 8-10 explants, P ROCK signaling with fasudil reduced photoreceptor degeneration and preserved the rod-bipolar synapse after retinal detachment. These results support the possibility, previously tested with Y27632, that ROCK inhibition may attenuate synaptic damage in iatrogenic detachments.

  18. Control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium, and l-cis-diltiazem.

    Science.gov (United States)

    Stern, J H; Kaupp, U B; MacLeish, P R

    1986-02-01

    The effect of calcium ions on the cGMP-activated current of outer segment membrane was examined by the excised-patch technique. Changes in the extracellular calcium concentration had marked effects on the cGMP-activated current, while changes in intracellular calcium concentration were ineffective. Changes in calcium concentration in the absence of cGMP had little, if any, effect on membrane conductance. These results suggest that both intracellular cGMP and extracellular calcium can directly affect the conductance underlying the light response in rod cells. The pharmacological agent l-cis-diltiazem reversibly inhibited the cGMP-activated current when applied to the intracellular side of an excised patch. When superfused over intact rod cells, l-cis-diltiazem reversibly blocked much of the normal light response. The isomer, d-cis-diltiazem, did not significantly affect either patches or intact rod cells. Thus, the light-regulated conductance has binding sites for both calcium and cGMP that may interact during the normal light response in rod cells and a site specific for l-cis-diltiazem that can be used to identify and further study the conductance mechanism.

  19. Multiple rod–cone and cone–rod photoreceptor transmutations in snakes: Evidence from visual opsin gene expression

    Science.gov (United States)

    Simoe, Bruno F; Sampaio, Filipa L.; Loew, Ellis R.; Sanders, Kate L.; Fisher, Robert N.; Hart, Nathan S.; Hunt, David M.; Partridge, Julian C.; Gower, David J.

    2016-01-01

    In 1934, Gordon Walls forwarded his radical theory of retinal photoreceptor ‘transmutation’. This proposed that rods and cones used for scotopic and photopic vision, respectively, were not fixed but could evolve into each other via a series of morphologically distinguishable intermediates. Walls' prime evidence came from series of diurnal and nocturnal geckos and snakes that appeared to have pure-cone or pure-rod retinas (in forms that Walls believed evolved from ancestors with the reverse complement) or which possessed intermediate photoreceptor cells. Walls was limited in testing his theory because the precise identity of visual pigments present in photoreceptors was then unknown. Subsequent molecular research has hitherto neglected this topic but presents new opportunities. We identify three visual opsin genes, rh1, sws1 and lws, in retinal mRNA of an ecologically and taxonomically diverse sample of snakes central to Walls' theory. We conclude that photoreceptors with superficially rod- or cone-like morphology are not limited to containing scotopic or photopic opsins, respectively. Walls' theory is essentially correct, and more research is needed to identify the patterns, processes and functional implications of transmutation. Future research will help to clarify the fundamental properties and physiology of photoreceptors adapted to function in different light levels.

  20. Ca sup 2+ binding capacity of cytoplasmic proteins from rod photoreceptors is mainly due to arrestin

    Energy Technology Data Exchange (ETDEWEB)

    Huppertz, B.; Weyand, I.; Bauer, P.J. (Institut fuer Biologische Informationsverarbeitung, Forschungszentrum Juelich GmbH (Germany, F.R.))

    1990-06-05

    Arrestin (also called S-antigen or 48-kDa protein) binds to photoexcited and phosphorylated rhodopsin and, thereby, blocks competitively the activation of transducin. Using Ca{sup 2+} titration in the presence of the indicator arsenazo III and {sup 45}Ca{sup 2+} autoradiography, we show that arrestin is a Ca2(+)-binding protein. The Ca{sup 2+} binding capacity of arresting-containing protein extracts from bovine rod outer segments is about twice as high as that of arrestin-depleted extracts. The difference in the Ca{sup 2+} binding of arrestin-containing and arrestin-depleted protein extracts was attributed to arrestin. Both, these difference-measurements of protein extracts and the measurements of purified arrestin yield dissociation constants for the Ca{sup 2+} binding of arrestin between 2 and 4 microM. The titration curves are consistent with a molar ratio of one Ca{sup 2+} binding site per arrestin. No Ca{sup 2+} binding in the micromolar range was found in extracts containing mainly transducin and cGMP-phosphodiesterase. Since arrestin is one of the most abundant proteins in rod photoreceptors occurring presumably up to millimolar concentrations in rod outer segments, we suggest that aside from its function to prevent the activation of transducin, arrestin acts probably as an intracellular Ca{sup 2+} buffer.

  1. Weak endogenous Ca2+ buffering supports sustained synaptic transmission by distinct mechanisms in rod and cone photoreceptors in salamander retina.

    Science.gov (United States)

    Van Hook, Matthew J; Thoreson, Wallace B

    2015-09-01

    Differences in synaptic transmission between rod and cone photoreceptors contribute to different response kinetics in rod- versus cone-dominated visual pathways. We examined Ca(2+) dynamics in synaptic terminals of tiger salamander photoreceptors under conditions that mimicked endogenous buffering to determine the influence on kinetically and mechanistically distinct components of synaptic transmission. Measurements of IC l(Ca) confirmed that endogenous Ca(2+) buffering is equivalent to ~0.05 mmol/L EGTA in rod and cone terminals. Confocal imaging showed that with such buffering, depolarization stimulated large, spatially unconstrained [Ca(2+)] increases that spread throughout photoreceptor terminals. We calculated immediately releasable pool (IRP) size and release efficiency in rods by deconvolving excitatory postsynaptic currents and presynaptic Ca(2+) currents. Peak efficiency of ~0.2 vesicles/channel was similar to that of cones (~0.3 vesicles/channel). Efficiency in both cell types was not significantly affected by using weak endogenous Ca(2+) buffering. However, weak Ca(2+) buffering speeded Ca(2+)/calmodulin (CaM)-dependent replenishment of vesicles to ribbons in both rods and cones, thereby enhancing sustained release. In rods, weak Ca(2+) buffering also amplified sustained release by enhancing CICR and CICR-stimulated release of vesicles at nonribbon sites. By contrast, elevating [Ca(2+)] at nonribbon sites in cones with weak Ca(2+) buffering and by inhibiting Ca(2+) extrusion did not trigger additional release, consistent with the notion that exocytosis from cones occurs exclusively at ribbons. The presence of weak endogenous Ca(2+) buffering in rods and cones facilitates slow, sustained exocytosis by enhancing Ca(2+)/CaM-dependent replenishment of ribbons in both rods and cones and by stimulating nonribbon release triggered by CICR in rods.

  2. Knocking Down Snrnp200 Initiates Demorphogenesis of Rod Photoreceptors in Zebrafish.

    Science.gov (United States)

    Liu, Yuan; Chen, Xue; Qin, Bing; Zhao, Kanxing; Zhao, Qingshun; Staley, Jonathan P; Zhao, Chen

    2015-01-01

    Purpose. The small nuclear ribonucleoprotein 200 kDa (SNRNP200) gene is a fundamental component for precursor message RNA (pre-mRNA) splicing and has been implicated in the etiology of autosomal dominant retinitis pigmentosa (adRP). This study aims to determine the consequences of knocking down Snrnp200 in zebrafish. Methods. Expression of the Snrnp200 transcript in zebrafish was determined via whole mount in situ hybridization. Morpholino oligonucleotide (MO) aiming to knock down the expression of Snrnp200 was injected into zebrafish embryos, followed by analyses of aberrant splicing and expression of the U4/U6-U5 tri-small nuclear ribonucleoproteins (snRNPs) components and retina-specific transcripts. Systemic changes and retinal phenotypes were further characterized by histological study and immunofluorescence staining. Results. Snrnp200 was ubiquitously expressed in zebrafish. Knocking down Snrnp200 in zebrafish triggered aberrant splicing of the cbln1 gene, upregulation of other U4/U6-U5 tri-snRNP components, and downregulation of a panel of retina-specific transcripts. Systemic defects were found correlated with knockdown of Snrnp200 in zebrafish. Only demorphogenesis of rod photoreceptors was detected in the initial stage, mimicking the disease characteristics of RP. Conclusions. We conclude that knocking down Snrnp200 in zebrafish could alter regular splicing and expression of a panel of genes, which may eventually trigger rod defects.

  3. Knocking Down Snrnp200 Initiates Demorphogenesis of Rod Photoreceptors in Zebrafish

    Directory of Open Access Journals (Sweden)

    Yuan Liu

    2015-01-01

    Full Text Available Purpose. The small nuclear ribonucleoprotein 200 kDa (SNRNP200 gene is a fundamental component for precursor message RNA (pre-mRNA splicing and has been implicated in the etiology of autosomal dominant retinitis pigmentosa (adRP. This study aims to determine the consequences of knocking down Snrnp200 in zebrafish. Methods. Expression of the Snrnp200 transcript in zebrafish was determined via whole mount in situ hybridization. Morpholino oligonucleotide (MO aiming to knock down the expression of Snrnp200 was injected into zebrafish embryos, followed by analyses of aberrant splicing and expression of the U4/U6-U5 tri-small nuclear ribonucleoproteins (snRNPs components and retina-specific transcripts. Systemic changes and retinal phenotypes were further characterized by histological study and immunofluorescence staining. Results. Snrnp200 was ubiquitously expressed in zebrafish. Knocking down Snrnp200 in zebrafish triggered aberrant splicing of the cbln1 gene, upregulation of other U4/U6-U5 tri-snRNP components, and downregulation of a panel of retina-specific transcripts. Systemic defects were found correlated with knockdown of Snrnp200 in zebrafish. Only demorphogenesis of rod photoreceptors was detected in the initial stage, mimicking the disease characteristics of RP. Conclusions. We conclude that knocking down Snrnp200 in zebrafish could alter regular splicing and expression of a panel of genes, which may eventually trigger rod defects.

  4. Flash responses of mouse rod photoreceptors in the isolated retina and corneal electroretinogram: comparison of gain and kinetics.

    Science.gov (United States)

    Heikkinen, Hanna; Vinberg, Frans; Pitkänen, Marja; Kommonen, Bertel; Koskelainen, Ari

    2012-08-17

    To examine the amplification and kinetics of murine rod photoresponses by recording ERG flash responses in vivo and ex vivo from the same retina. We also aimed to evaluate the two available methods for isolating the rod signal from the ERG flash response, that is, pharmacology and paired flash method on the isolated retina. Dark-adapted ERG responses to full-field flashes of green light were recorded from anesthetized (ketamine/xylazine) C57BL/6N mice. ERG flash responses to homogenous light stimuli arriving from the photoreceptor side were then recorded transretinally from the same retinas, isolated and perfused with Ringer's or Ames' solution at 37°C. The responses were analyzed to determine the a-wave kinetics as well as the estimated flash sensitivity and kinetics of the full rod responses derived with the paired flash protocol. The analysis was complemented with pharmacologic blockade of glutamatergic transmission in the isolated retina. The a-waves were of comparable size, sensitivity and kinetics in vivo and in the isolated retina, but the onset of the b-wave was delayed in the isolated retina. The Lamb-Pugh activation constants determined for the a-waves were similar in both preparations. The kinetics of the derived photoreceptor responses were similar in both conditions, although the responses were consistently slightly slower ex vivo. This was not explicable as a direct effect of ketamine or xylazine on the photoreceptors or as their indirect effect through hyperglycemia, as tested on the isolated retina. Through comparison to the corneal ERG, the transretinal ERG is a valuable tool for assaying the physiologic state of isolated retinal tissue. The rod photoreceptor responses of the intact isolated retina correspond well to those recorded in vivo. The origin of their faster kinetics compared to single cell recordings remains to be determined.

  5. Feedback Induction of a Photoreceptor-specific Isoform of Retinoid-related Orphan Nuclear Receptor β by the Rod Transcription Factor NRL*

    Science.gov (United States)

    Fu, Yulong; Liu, Hong; Ng, Lily; Kim, Jung-Woong; Hao, Hong; Swaroop, Anand; Forrest, Douglas

    2014-01-01

    Vision requires the generation of cone and rod photoreceptors that function in daylight and dim light, respectively. The neural retina leucine zipper factor (NRL) transcription factor critically controls photoreceptor fates as it stimulates rod differentiation and suppresses cone differentiation. However, the controls over NRL induction that balance rod and cone fates remain unclear. We have reported previously that the retinoid-related orphan receptor β gene (Rorb) is required for Nrl expression and other retinal functions. We show that Rorb differentially expresses two isoforms: RORβ2 in photoreceptors and RORβ1 in photoreceptors, progenitor cells, and other cell types. Deletion of RORβ2 or RORβ1 increased the cone:rod ratio ∼2-fold, whereas deletion of both isoforms in Rorb−/− mice produced almost exclusively cone-like cells at the expense of rods, suggesting that both isoforms induce Nrl. Electroporation of either RORβ isoform into retinal explants from Rorb−/− neonates reactivated Nrl and rod genes but, in Nrl−/− explants, failed to reactivate rod genes, indicating that NRL is the effector for both RORβ isoforms in rod differentiation. Unexpectedly, RORβ2 expression was lost in Nrl−/− mice. Moreover, NRL activated the RORβ2-specific promoter of Rorb, indicating that NRL activates Rorb, its own inducer gene. We suggest that feedback activation between Nrl and Rorb genes reinforces the commitment to rod differentiation. PMID:25296752

  6. Feedback induction of a photoreceptor-specific isoform of retinoid-related orphan nuclear receptor β by the rod transcription factor NRL.

    Science.gov (United States)

    Fu, Yulong; Liu, Hong; Ng, Lily; Kim, Jung-Woong; Hao, Hong; Swaroop, Anand; Forrest, Douglas

    2014-11-21

    Vision requires the generation of cone and rod photoreceptors that function in daylight and dim light, respectively. The neural retina leucine zipper factor (NRL) transcription factor critically controls photoreceptor fates as it stimulates rod differentiation and suppresses cone differentiation. However, the controls over NRL induction that balance rod and cone fates remain unclear. We have reported previously that the retinoid-related orphan receptor β gene (Rorb) is required for Nrl expression and other retinal functions. We show that Rorb differentially expresses two isoforms: RORβ2 in photoreceptors and RORβ1 in photoreceptors, progenitor cells, and other cell types. Deletion of RORβ2 or RORβ1 increased the cone:rod ratio ∼2-fold, whereas deletion of both isoforms in Rorb(-/-) mice produced almost exclusively cone-like cells at the expense of rods, suggesting that both isoforms induce Nrl. Electroporation of either RORβ isoform into retinal explants from Rorb(-/-) neonates reactivated Nrl and rod genes but, in Nrl(-/-) explants, failed to reactivate rod genes, indicating that NRL is the effector for both RORβ isoforms in rod differentiation. Unexpectedly, RORβ2 expression was lost in Nrl(-/-) mice. Moreover, NRL activated the RORβ2-specific promoter of Rorb, indicating that NRL activates Rorb, its own inducer gene. We suggest that feedback activation between Nrl and Rorb genes reinforces the commitment to rod differentiation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. A hybrid photoreceptor expressing both rod and cone genes in a mouse model of enhanced S-cone syndrome.

    Directory of Open Access Journals (Sweden)

    Joseph C Corbo

    2005-08-01

    Full Text Available Rod and cone photoreceptors subserve vision under dim and bright light conditions, respectively. The differences in their function are thought to stem from their different gene expression patterns, morphologies, and synaptic connectivities. In this study, we have examined the photoreceptor cells of the retinal degeneration 7(rd7 mutant mouse, a model for the human enhanced S-cone syndrome (ESCS. This mutant carries a spontaneous deletion in the mouse ortholog of NR2E3, an orphan nuclear receptor transcription factor mutated in ESCS. Employing microarray and in situ hybridization analysis we have found that the rd7 retina contains a modestly increased number of S-opsin-expressing cells that ultrastructurally appear to be normal cones. Strikingly, the majority of the photoreceptors in the rd7 retina represent a morphologically hybrid cell type that expresses both rod- and cone-specific genes. In addition, in situ hybridization screening of genes shown to be up-regulated in the rd7 mutant retina by microarray identified ten new cone-specific or cone-enriched genes with a wide range of biochemical functions, including two genes specifically involved in glucose/glycogen metabolism. We suggest that the abnormal electroretinograms, slow retinal degeneration, and retinal dysmorphology seen in humans with ESCS may, in part, be attributable to the aberrant function of a hybrid photoreceptor cell type similar to that identified in this study. The functional diversity of the novel cone-specific genes identified here indicates molecular differences between rods and cones extending far beyond those previously discovered.

  8. Growth Factor Receptor-Bound Protein 14 Undergoes Light-Dependent Intracellular Translocation in Rod Photoreceptors: Functional Role on Retinal Insulin Receptor Activation

    OpenAIRE

    Rajala, Ammaji; Roger J. Daly; Tanito, Masaki; Allen, Dustin T.; Lowenna J Holt; Lobanova, Ekaterina; Arshavsky, Vadim Y; Rajala, Raju V.S.

    2009-01-01

    Growth factor receptor-bound protein 14 (Grb14) is involved in growth factor receptor tyrosine kinase signaling. Here we report that light causes a major redistribution of Grb14 among the individual subcellular compartments of the retinal rod photoreceptor. Grb14 is localized predominantly to the inner segment, nuclear layer and synapse in dark-adapted rods, whereas in the light-adapted rods, Grb14 redistributed throughout the entire cell, including the outer segment. The translocation of Grb...

  9. Human retinal disease from AIPL1 gene mutations: foveal cone loss with minimal macular photoreceptors and rod function remaining.

    Science.gov (United States)

    Jacobson, Samuel G; Cideciyan, Artur V; Aleman, Tomas S; Sumaroka, Alexander; Roman, Alejandro J; Swider, Malgorzata; Schwartz, Sharon B; Banin, Eyal; Stone, Edwin M

    2011-01-05

    To determine the human retinal phenotype caused by mutations in the gene encoding AIPL1 (Aryl hydrocarbon receptor-interacting protein-like 1) now that there are proof-of-concept results for gene therapy success in Aipl1-deficient mice. Leber congenital amaurosis (LCA) patients (n = 10) and one patient with a later-onset retinal degeneration (RD) and AIPL1 mutations were studied by ocular examination, retinal imaging, perimetry, full-field sensitivity testing, and pupillometry. The LCA patients had severe visual acuity loss early in life, nondetectable electroretinograms (ERGs), and little or no detectable visual fields. Hallmarks of retinal degeneration were present in a wide region, including the macula and midperiphery; there was some apparent peripheral retinal sparing. Cross-sectional imaging showed foveal cone photoreceptor loss with a ring of minimally preserved paracentral photoreceptor nuclear layer. Features of retinal remodeling were present eccentric to the region of detectable photoreceptors. Full-field sensitivity was reduced by at least 2 log units, and chromatic stimuli, by psychophysics and pupillometry, revealed retained but impaired rod function. The RD patient, examined serially over two decades (ages, 45-67 years), retained an ERG in the fifth decade of life with abnormal rod and cone signals; and there was progressive loss of central and peripheral function. AIPL1-LCA, unlike some other forms of LCA with equally severe visual disturbance, shows profound loss of foveal as well as extrafoveal photoreceptors. The more unusual late-onset and slower form of AIPL1 disease may be better suited to gene augmentation therapy and is worthy of detection and further study.

  10. Rod and cone photoreceptor cells produce ROS in response to stress in a live retinal explant system.

    LENUS (Irish Health Repository)

    Bhatt, Lavinia

    2010-01-01

    PURPOSE: The production of reactive oxygen species (ROS) can lead to oxidative stress, which is a strong contributory factor to many ocular diseases. In this study, the removal of trophic factors is used as a model system to investigate the effects of stress in the retina. The aims were to determine if both rod and cone photoreceptor cells produce ROS when they are deprived of trophic factor support and to demonstrate if the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes are responsible for this ROS production. METHODS: Retinas were explanted from mice aged between postnatal days 8-10 and cultured overnight. The following morning, confocal microscopy combined with various fluorescent probes was used to detect the production of ROS. Each time peanut agglutinin (PNA), a cone photoreceptor marker, was used to facilitate orientation of the retina. Dihydroethidium and dihydrorhodamine 123 (DHR123) were used to determine which cells produce ROS. Subsequently, western blots of retinal serial sections were used to detect the presence of Noxs in the different retinal layers. The Nox inhibitor apocynin was then tested to determine if it altered the production of ROS within these cells. RESULTS: Live retinal explants, viewed at high magnifications using confocal microscopy, displayed an increase in the fluorescent products of dihydroethidium and DHR123 upon serum removal when compared to controls. DHR123 fluorescence, once oxidized, localized to mitochondria and was found in the same focal plane as the PNA staining. This showed that cones and rods produced ROS when stressed. Retinal serial sectioning established that the photoreceptor layer expressed Nox4, dual oxidase (Duox) 1, and Duox2 at varying levels. Finally, the Nox inhibitor apocynin decreased the burst stimulated by the stress of serum removal. CONCLUSIONS: Confocal microscopy and PNA staining allowed differentiation of cell types within the outermost layers of the retina, demonstrating

  11. Rapid kinetics of endocytosis at rod photoreceptor synapses depends upon endocytic load and calcium.

    Science.gov (United States)

    Cork, Karlene M; Thoreson, Wallace B

    2014-05-01

    Release from rods is triggered by the opening of L-type Ca2+ channels that lie beneath synaptic ribbons. After exocytosis, vesicles are retrieved by compensatory endocytosis. Previous work showed that endocytosis is dynamin-dependent in rods but dynamin-independent in cones. We hypothesized that fast endocytosis in rods may also differ from cones in its dependence upon the amount of Ca2+ influx and/or endocytic load. We measured exocytosis and endocytosis from membrane capacitance (C m) changes evoked by depolarizing steps in voltage clamped rods from tiger salamander retinal slices. Similar to cones, the time constant for endocytosis in rods was quite fast, averaging endocytosis kinetics in rods slowed after increasing Ca2+ channel activation with longer step durations or more strongly depolarized voltage steps. Endocytosis kinetics also slowed as Ca2+ buffering was decreased by replacing BAPTA (10 or 1 mM) with the slower Ca2+ buffer EGTA (5 or 0.5 mM) in the pipette solution. These data provide further evidence that endocytosis mechanisms differ in rods and cones and suggest that endocytosis in rods is regulated by both endocytic load and local Ca2+ levels.

  12. Protein and signaling networks in vertebrate photoreceptor cells

    Directory of Open Access Journals (Sweden)

    Karl-Wilhelm eKoch

    2015-11-01

    Full Text Available Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cGMP and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase GRK1 under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases and is regulated by specific neuronal Ca2+-sensor proteins called GCAPs. At least one guanylate cyclase (ROS-GC1 was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments.

  13. Rod differentiation factor NRL activates the expression of nuclear receptor NR2E3 to suppress the development of cone photoreceptors.

    Science.gov (United States)

    Oh, Edwin C T; Cheng, Hong; Hao, Hong; Jia, Lin; Khan, Naheed Wali; Swaroop, Anand

    2008-10-21

    Neural developmental programs require a high level of coordination between the decision to exit cell cycle and acquisition of cell fate. The Maf-family transcription factor NRL is essential for rod photoreceptor specification in the mammalian retina as its loss of function converts rod precursors to functional cones. Ectopic expression of NRL or a photoreceptor-specific orphan nuclear receptor NR2E3 completely suppresses cone development while concurrently directing the post-mitotic photoreceptor precursors towards rod cell fate. Given that NRL and NR2E3 have overlapping functions and NR2E3 expression is abolished in the Nrl(-/-) retina, we wanted to clarify the distinct roles of NRL and NR2E3 during retinal differentiation. Here, we demonstrate that NRL binds to a sequence element in the Nr2e3 promoter and enhances its activity synergistically with the homeodomain protein CRX. Using transgenic mice, we show that NRL can only partially suppress cone development in the absence of NR2E3. Gene profiling of retinas from transgenic mice that ectopically express NR2E3 or NRL in cone precursors reveals overlapping and unique targets of these two transcription factors. Together with previous reports, our findings establish the hierarchy of transcriptional regulators in determining rod versus cone cell fate in photoreceptor precursors during the development of mammalian retina.

  14. Nrl-Cre transgenic mouse mediates loxP recombination in developing rod photoreceptors.

    Science.gov (United States)

    Brightman, Diana S; Razafsky, David; Potter, Chloe; Hodzic, Didier; Chen, Shiming

    2016-03-01

    The developing mouse retina is a tractable model for studying neurogenesis and differentiation. Although transgenic Cre mouse lines exist to mediate conditional genetic manipulations in developing mouse retinas, none of them act specifically in early developing rods. For conditional genetic manipulations of developing retinas, a Nrl-Cre mouse line in which the Nrl promoter drives expression of Cre in rod precursors was created. The results showed that Nrl-Cre expression was specific to the retina where it drives rod-specific recombination with a temporal pattern similar to endogenous Nrl expression during retinal development. This Nrl-Cre transgene does not negatively impact retinal structure and function. Taken together, the data suggested that the Nrl-Cre mouse line was a valuable tool to drive Cre-mediated recombination specifically in developing rods. © 2016 Wiley Periodicals, Inc.

  15. Pias3 is necessary for dorso-ventral patterning and visual response of retinal cones but is not required for rod photoreceptor differentiation

    Directory of Open Access Journals (Sweden)

    Christie K. Campla

    2017-06-01

    Full Text Available Protein inhibitor of activated Stat 3 (Pias3 is implicated in guiding specification of rod and cone photoreceptors through post-translational modification of key retinal transcription factors. To investigate its role during retinal development, we deleted exon 2-5 of the mouse Pias3 gene, which resulted in complete loss of the Pias3 protein. Pias3−/− mice did not show any overt phenotype, and retinal lamination appeared normal even at 18 months. We detected reduced photopic b-wave amplitude by electroretinography following green light stimulation of postnatal day (P21 Pias3−/− retina, suggesting a compromised visual response of medium wavelength (M cones. No change was evident in response of short wavelength (S cones or rod photoreceptors until 7 months. Increased S-opsin expression in the M-cone dominant dorsal retina suggested altered distribution of cone photoreceptors. Transcriptome profiling of P21 and 18-month-old Pias3−/− retina revealed aberrant expression of a subset of photoreceptor genes. Our studies demonstrate functional redundancy in SUMOylation-associated transcriptional control mechanisms and identify a specific, though limited, role of Pias3 in modulating spatial patterning and optimal function of cone photoreceptor subtypes in the mouse retina.

  16. Loss of retinoschisin (RS1) cell surface protein in maturing mouse rod photoreceptors elevates the luminance threshold for light-driven translocation of transducin but not arrestin.

    Science.gov (United States)

    Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bush, Ronald A; Sieving, Paul A

    2012-09-19

    Loss of retinoschisin (RS1) in Rs1 knock-out (Rs1-KO) retina produces a post-photoreceptor phenotype similar to X-linked retinoschisis in young males. However, Rs1 is expressed strongly in photoreceptors, and Rs1-KO mice have early reduction in the electroretinogram a-wave. We examined light-activated transducin and arrestin translocation in young Rs1-KO mice as a marker for functional abnormalities in maturing rod photoreceptors. We found a progressive reduction in luminance threshold for transducin translocation in wild-type (WT) retinas between postnatal days P18 and P60. At P21, the threshold in Rs1-KO retinas was 10-fold higher than WT, but it decreased to translocation and re-translocation of transducin in the dark were not affected. Rs1-KO rod outer segment (ROS) length was significantly shorter than WT at P21 but was comparable with WT at P60. These findings suggested a delay in the structural and functional maturation of Rs1-KO ROS. Consistent with this, transcription factors CRX and NRL, which are fundamental to maturation of rod protein expression, were reduced in ROS of Rs1-KO mice at P21 but not at P60. Expression of transducin was 15-30% lower in P21 Rs1-KO ROS and transducin GTPase hydrolysis was nearly twofold faster, reflecting a 1.7- to 2.5-fold increase in RGS9 (regulator of G-protein signaling) level. Transduction protein expression and activity levels were similar to WT at P60. Transducin translocation threshold elevation indicates photoreceptor functional abnormalities in young Rs1-KO mice. Rapid reduction in threshold coupled with age-related changes in transduction protein levels and transcription factor expression are consistent with delayed maturation of Rs1-KO photoreceptors.

  17. Transcriptome Dynamics of Developing Photoreceptors in Three-Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks.

    Science.gov (United States)

    Kaewkhaw, Rossukon; Kaya, Koray Dogan; Brooks, Matthew; Homma, Kohei; Zou, Jizhong; Chaitankar, Vijender; Rao, Mahendra; Swaroop, Anand

    2015-12-01

    The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile, by RNA-seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures.

  18. Neurotoxicity of cGMP in the vertebrate retina: from the initial research on rd mutant mice to zebrafish genetic approaches.

    Science.gov (United States)

    Iribarne, Maria; Masai, Ichiro

    2017-09-01

    Zebrafish are an excellent animal model for research on vertebrate development and human diseases. Sophisticated genetic tools including large-scale mutagenesis methodology make zebrafish useful for studying neuronal degenerative diseases. Here, we review zebrafish models of inherited ophthalmic diseases, focusing on cGMP metabolism in photoreceptors. cGMP is the second messenger of phototransduction, and abnormal cGMP levels are associated with photoreceptor death. cGMP concentration represents a balance between cGMP phosphodiesterase 6 (PDE6) and guanylate cyclase (GC) activities in photoreceptors. Various zebrafish cGMP metabolism mutants were used to clarify molecular mechanisms by which dysfunctions in this pathway trigger photoreceptor degeneration. Here, we review the history of research on the retinal degeneration (rd) mutant mouse, which carries a genetic mutation of PDE6b, and we also highlight recent research in photoreceptor degeneration using zebrafish models. Several recent discoveries that provide insight into cGMP toxicity in photoreceptors are discussed.

  19. The effect of recombinant recoverin on the photoresponse of truncated rod photoreceptors

    Science.gov (United States)

    Erickson, Martha A.; Lagnado, Leon; Zozulya, Sergey; Neubert, Thomas A.; Stryer, Lubert; Baylor, Denis A.

    1998-01-01

    Recoverin is a heterogeneously acylated calcium-binding protein thought to regulate visual transduction. Its effect on the photoresponse was investigated by dialyzing the recombinant protein into truncated salamander rod outer segments. At high Ca2+ (Ca), myristoylated recoverin (Ca-recoverin) prolonged the recovery phase of the bright flash response but had less effect on the dim flash response. The prolongation of recovery had an apparent Kd for Ca of 13 μM and a Hill coefficient of 2. The prolongation was shown to be mediated by inhibition of rhodopsin deactivation. After a sudden imposed drop in Ca concentration, the effect of recoverin switched off with little lag. The myristoyl (C14:0) modification of recoverin increased its activity 12-fold, and the C12:0 or C14:2 acyl group gave similar effects. These experiments support the notion that recoverin mediates Ca-dependent inhibition of rhodopsin phosphorylation and thereby controls light-triggered phosphodiesterase activity, particularly at high light levels. PMID:9600991

  20. Two types of Tet-On transgenic lines for doxycycline-inducible gene expression in zebrafish rod photoreceptors and a gateway-based tet-on toolkit.

    Directory of Open Access Journals (Sweden)

    Leah J Campbell

    Full Text Available The ability to control transgene expression within specific tissues is an important tool for studying the molecular and cellular mechanisms of development, physiology, and disease. We developed a Tet-On system for spatial and temporal control of transgene expression in zebrafish rod photoreceptors. We generated two transgenic lines using the Xenopus rhodopsin promoter to drive the reverse tetracycline-controlled transcriptional transactivator (rtTA, one with self-reporting GFP activity and one with an epitope tagged rtTA. The self-reporting line includes a tetracycline response element (TRE-driven GFP and, in the presence of doxycycline, expresses GFP in larval and adult rods. A time-course of doxycycline treatment demonstrates that maximal induction of GFP expression, as determined by the number of GFP-positive rods, is reached within approximately 24 hours of drug treatment. The epitope-tagged transgenic line eliminates the need for the self-reporting GFP activity by expressing a FLAG-tagged rtTA protein. Both lines demonstrate strong induction of TRE-driven transgenes from plasmids microinjected into one-cell embryos. These results show that spatial and temporal control of transgene expression can be achieved in rod photoreceptors. Additionally, system components are constructed in Gateway compatible vectors for the rapid cloning of doxycycline-inducible transgenes and use in other areas of zebrafish research.

  1. Regulation of a novel isoform of Receptor Expression Enhancing Protein REEP6 in rod photoreceptors by bZIP transcription factor NRL.

    Science.gov (United States)

    Hao, Hong; Veleri, Shobi; Sun, Bo; Kim, Douglas S; Keeley, Patrick W; Kim, Jung-Woong; Yang, Hyun-Jin; Yadav, Sharda P; Manjunath, Souparnika H; Sood, Raman; Liu, Paul; Reese, Benjamin E; Swaroop, Anand

    2014-08-15

    The Maf-family leucine zipper transcription factor NRL is essential for rod photoreceptor development and functional maintenance in the mammalian retina. Mutations in NRL are associated with human retinopathies, and loss of Nrl in mice leads to a cone-only retina with the complete absence of rods. Among the highly down-regulated genes in the Nrl(-/-) retina, we identified receptor expression enhancing protein 6 (Reep6), which encodes a member of a family of proteins involved in shaping of membrane tubules and transport of G-protein coupled receptors. Here, we demonstrate the expression of a novel Reep6 isoform (termed Reep6.1) in the retina by exon-specific Taqman assay and rapid analysis of complementary deoxyribonucleic acid (cDNA) ends (5'-RACE). The REEP6.1 protein includes 27 additional amino acids encoded by exon 5 and is specifically expressed in rod photoreceptors of developing and mature retina. Chromatin immunoprecipitation assay identified NRL binding within the Reep6 intron 1. Reporter assays in cultured cells and transfections in retinal explants mapped an intronic enhancer sequence that mediated NRL-directed Reep6.1 expression. We also demonstrate that knockdown of Reep6 in mouse and zebrafish resulted in death of retinal cells. Our studies implicate REEP6.1 as a key functional target of NRL-centered transcriptional regulatory network in rod photoreceptors. Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  2. Lack of effect of microfilament or microtubule cytoskeleton-disrupting agents on restriction of externalized phosphatidylserine to rod photoreceptor outer segment tips.

    Science.gov (United States)

    Ruggiero, Linda; Finnemann, Silvia C

    2014-01-01

    In the mammalian retina, life-long renewal of rod photoreceptor outer segments involves circadian shedding of distal outer segment tips and their prompt phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning after light onset. Failure of this process causes retinal dystrophy in animal models and its decline likely contributes to retinal aging and some forms of degeneration of the human retina. We previously found that surface exposure of the membrane phospholipid phosphatidylserine (PS) is restricted to outer segment tips with discrete boundaries in mouse retina and that both frequency and length of tips exposing PS peak after light onset. Here, we sought to test mechanisms photoreceptors use to restrict PS specifically to their outer segment tips. To this end, we tested whether nocodazole or cytochalasin D, perturbing microtubule or F-actin microfilament cytoskeleton, respectively, affect localization of externalized PS at outer segment tips. Fluorescence imaging of PS exposed by rods in freshly dissected, live mouse retina showed normal PS demarcation of outer segment tips regardless of drug treatment. These results suggest that the mechanism that restricts externalized PS to rod tips is independent of F-actin and microtubule cytoskeletal systems.

  3. STEREOLOGY AND SOME STRUCTURAL CORRELATES OF RETINAL AND PHOTORECEPTOR CELL FUNCTION

    Directory of Open Access Journals (Sweden)

    Terry M Mayhew

    2011-05-01

    Full Text Available The retina is the part of the eye which detects light, transduces it into nerve impulses and plays a significant role in visual perception. Sensitivity to light is multi-factorial and depends on the properties of photopigment molecules, their synthesis and incorporation into photoreceptor membranes and the neural circuitry between photoreceptor cells, bipolar neurons and ganglion neurons. In addition, it depends on structural factors such as the absolute and relative numbers of different types of photoreceptor neurons, their subcellular morphology, their distribution across the retina and the physical dimensions (especially surface areas and spatial arrangements of their photoreceptor membranes. At the molecular level, these membranes harbour photosensitive pigment molecules comprising transmembrane glycoproteins (opsins, which vary between photoreceptor cells and a non-protein chromophore. Phototransduction involves a conformational change in the chromophore and activation of an opsin. A transducer G protein, transducin, lowers levels of cGMP and triggers changes in membrane ion permeability including the closure of Na+ channels. This causes the plasmalemma to become less depolarized and the relative hyperpolarization stimulates ganglion cells whose axons form the optic nerve. Phosducin is a light-regulated phosphoprotein located in inner and outer segments of rod photoreceptor cells. It modulates phototransduction by binding to beta and gamma subunits of transducin. This review briefly illustrates ways in which stereology can contribute to our understanding of these processes by providing quantitative data on photoreceptor number, disk membrane surface area and the subcellular immunolocalisation of key molecules.

  4. Mouse ganglion-cell photoreceptors are driven by the most sensitive rod pathway and by both types of cones.

    Directory of Open Access Journals (Sweden)

    Shijun Weng

    Full Text Available Intrinsically photosensitive retinal ganglion cells (iprgcs are depolarized by light by two mechanisms: directly, through activation of their photopigment melanopsin; and indirectly through synaptic circuits driven by rods and cones. To learn more about the rod and cone circuits driving ipRGCs, we made multielectrode array (MEA and patch-clamp recordings in wildtype and genetically modified mice. Rod-driven ON inputs to ipRGCs proved to be as sensitive as any reaching the conventional ganglion cells. These signals presumably pass in part through the primary rod pathway, involving rod bipolar cells and AII amacrine cells coupled to ON cone bipolar cells through gap junctions. Consistent with this interpretation, the sensitive rod ON input to ipRGCs was eliminated by pharmacological or genetic disruption of gap junctions, as previously reported for conventional ganglion cells. A presumptive cone input was also detectable as a brisk, synaptically mediated ON response that persisted after disruption of rod ON pathways. This was roughly three log units less sensitive than the rod input. Spectral analysis revealed that both types of cones, the M- and S-cones, contribute to this response and that both cone types drive ON responses. This contrasts with the blue-OFF, yellow-ON chromatic opponency reported in primate ipRGCs. The cone-mediated response was surprisingly persistent during steady illumination, echoing the tonic nature of both the rod input to ipRGCs and their intrinsic, melanopsin-based phototransduction. These synaptic inputs greatly expand the dynamic range and spectral bandpass of the non-image-forming visual functions for which ipRGCs provide the principal retinal input.

  5. Clinical characteristics of rod and cone photoreceptor dystrophies in patients with mutations in the C8orf37 gene

    NARCIS (Netherlands)

    Huet, R.A.C. van; Estrada-Cuzcano, A.; Banin, E.; Rotenstreich, Y.; Hipp, S.; Kohl, S.; Hoyng, C.B.; Hollander, A.I. den; Collin, R.W.J.; Klevering, B.J.

    2013-01-01

    PURPOSE: To provide the clinical features in patients with retinal disease caused by C8orf37 gene mutations. METHODS: Eight patients--four diagnosed with retinitis pigmentosa (RP) and four with cone-rod dystrophy (CRD), carrying causal C8orf37 mutations--were clinically evaluated, including

  6. Regulation of cyclic GMP metabolism in toad photoreceptors. Definition of the metabolic events subserving photoexcited and attenuated states.

    Science.gov (United States)

    Dawis, S M; Graeff, R M; Heyman, R A; Walseth, T F; Goldberg, N D

    1988-06-25

    Photoreceptor metabolism of cGMP and its regulation were characterized in isolated toad retinas by determining the intensity and time dependence of light-induced changes in the following metabolic parameters: cGMP hydrolytic flux determined by the rate of 18O incorporation from 18O-water into retinal guanine nucleotide alpha-phosphoryls; changes in the total (protein-bound and unbound) concentrations of the guanine nucleotide metabolic intermediates; and changes in the concentration of metabolic (unbound) GDP calculated from the fraction of the alpha-GDP that undergoes labeling with 18O. The latter is interpreted to reflect the state of the equilibrium between GDP- and GTP-complexed forms of G-protein. With narrow band 500 nm light that preferentially stimulates red rod photoreceptors, a range of intensities covering approximately 5 log units produced increases of over 10-fold in cGMP metabolic flux. However, the characteristics of the cGMP metabolic response over the first 2.5 log units of intensity are readily distinguishable from those at higher intensities which exhibit progressive attenuation by an intensity- and time-dependent process. Over the range of low intensities (0.6-3 log photons.micron-2.s-1) the metabolic response is characterized by 1) increases in cGMP hydrolytic flux of up to 8-fold as a logarithmic function of intensity of photic stimulation that are sustained for at least 200 s; 2) small increases or no change in the concentration of total cGMP; 3) large increases of up to 10-fold in the concentration of metabolically active GDP as a linear function of intensity with no significant change in the tissue concentrations of total GDP or GTP; and 4) amplification of the photosignal by the metabolism of approximately 10,000 molecules of cGMP per photoisomerization with the major site of amplification at the level of the interaction of bleached rhodopsin with G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Functional and Molecular Characterization of Rod-like Cells from Retinal Stem Cells Derived from the Adult Ciliary Epithelium

    Science.gov (United States)

    Demontis, Gian Carlo; Aruta, Claudia; Comitato, Antonella; De Marzo, Anna; Marigo, Valeria

    2012-01-01

    In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor features. Rod maturation was evaluated at two levels: gene expression and electrophysiological functionality. Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro. We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP. We also identified voltage-gated channels necessary for rod maturation and viability. This level of analysis for the first time provides evidence that adult retinal stem cells can generate highly homogeneous rod-fated cells. PMID:22432014

  8. Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium.

    Directory of Open Access Journals (Sweden)

    Gian Carlo Demontis

    Full Text Available In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor features. Rod maturation was evaluated at two levels: gene expression and electrophysiological functionality. Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro. We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP. We also identified voltage-gated channels necessary for rod maturation and viability. This level of analysis for the first time provides evidence that adult retinal stem cells can generate highly homogeneous rod-fated cells.

  9. Photoreceptor engineering

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    Thea eZiegler

    2015-06-01

    Full Text Available Sensory photoreceptors not only control diverse adaptive responses in Nature, but as light-regulated actuators they also provide the foundation for optogenetics, the non-invasive and spatiotemporally precise manipulation of cellular events by light. Novel photoreceptors have been engineered that establish control by light over manifold biological processes previously inaccessible to optogenetic intervention. Recently, photoreceptor engineering has witnessed a rapid development, and light-regulated actuators for the perturbation of a plethora of cellular events are now available. Here, we review fundamental principles of photoreceptors and light-regulated allostery. Photoreceptors dichotomize into associating receptors that alter their oligomeric state as part of light-regulated allostery and non-associating receptors that do not. A survey of engineered photoreceptors pinpoints light-regulated association reactions and order-disorder transitions as particularly powerful and versatile design principles. Photochromic photoreceptors that are bidirectionally toggled by two light colors augur enhanced spatiotemporal resolution and use as photoactivatable fluorophores. By identifying desirable traits in engineered photoreceptors, we provide pointers for the design of future, light-regulated actuators.

  10. Direct cell fate conversion of human somatic stem cells into cone and rod photoreceptor-like cells by inhibition of microRNA-203

    OpenAIRE

    Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Shin, Tae-Hoon; Seo, Yoojin; Kim, Hyung-Sik; Kang, Kyung-Sun

    2016-01-01

    Stem cell-based photoreceptor differentiation strategies have been the recent focus of therapies for retinal degenerative diseases. Previous studies utilized embryonic stem (ES) cells and neural retina differentiation cocktails, including DKK1 and Noggin. Here, we show a novel microRNA-mediated strategy of retina differentiation from somatic stem cells, which are potential allogeneic cell sources. Human amniotic epithelial stem cells (AESCs) and umbilical cord blood-derived mesenchymal stem c...

  11. Direct cell fate conversion of human somatic stem cells into cone and rod photoreceptor-like cells by inhibition of microRNA-203.

    Science.gov (United States)

    Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Shin, Tae-Hoon; Seo, Yoojin; Kim, Hyung-Sik; Kang, Kyung-Sun

    2016-07-05

    Stem cell-based photoreceptor differentiation strategies have been the recent focus of therapies for retinal degenerative diseases. Previous studies utilized embryonic stem (ES) cells and neural retina differentiation cocktails, including DKK1 and Noggin. Here, we show a novel microRNA-mediated strategy of retina differentiation from somatic stem cells, which are potential allogeneic cell sources. Human amniotic epithelial stem cells (AESCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) treated with a retina differentiation cocktail induced gene expressions of retina development-relevant genes. Furthermore, microRNA-203 (miR-203) is abundantly expressed in human AESCs and human UCB-MSCs. This miR-203 is predicted to target multiple retina development-relevant genes, particularly DKK1, CRX, RORβ, NEUROD1, NRL and THRB. The inhibition of miR-203 induced a retina differentiation of AESCs and UCB-MSCs. Moreover, successive treatments of anti-miR-203 led to the expression of both mature photoreceptor (PR) markers, rhodopsin and opsin. In addition, we determined that CRX, NRL and DKK1 are direct targets of miR-203 using a luciferase assay. Thus, the work presented here suggests that somatic stem cells can potentially differentiate into neural retina cell types when treated with anti-miR-203. They may prove to be a source of both PR subtypes for future allogeneic stem cell-based therapies of non-regenerative retina diseases.

  12. The role of mislocalized phototransduction in photoreceptor cell death of retinitis pigmentosa.

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    Takeshi Nakao

    Full Text Available Most of inherited retinal diseases such as retinitis pigmentosa (RP cause photoreceptor cell death resulting in blindness. RP is a large family of diseases in which the photoreceptor cell death can be caused by a number of pathways. Among them, light exposure has been reported to induce photoreceptor cell death. However, the detailed mechanism by which photoreceptor cell death is caused by light exposure is unclear. In this study, we have shown that even a mild light exposure can induce ectopic phototransduction and result in the acceleration of rod photoreceptor cell death in some vertebrate models. In ovl, a zebrafish model of outer segment deficiency, photoreceptor cell death is associated with light exposure. The ovl larvae show ectopic accumulation of rhodopsin and knockdown of ectopic rhodopsin and transducin rescue rod photoreceptor cell death. However, knockdown of phosphodiesterase, the enzyme that mediates the next step of phototransduction, does not. So, ectopic phototransduction activated by light exposure, which leads to rod photoreceptor cell death, is through the action of transducin. Furthermore, we have demonstrated that forced activation of adenylyl cyclase in the inner segment leads to rod photoreceptor cell death. For further confirmation, we have also generated a transgenic fish which possesses a human rhodopsin mutation, Q344X. This fish and rd10 model mice show photoreceptor cell death caused by adenylyl cyclase. In short, our study indicates that in some RP, adenylyl cyclase is involved in photoreceptor cell death pathway; its inhibition is potentially a logical approach for a novel RP therapy.

  13. Transgenic Mice for cGMP Imaging

    Science.gov (United States)

    Thunemann, Martin; Wen, Lai; Hillenbrand, Matthias; Vachaviolos, Angelos; Feil, Susanne; Ott, Thomas; Han, Xiaoxing; Fukumura, Dai; Jain, Rakesh K.; Russwurm, Michael; de Wit, Cor; Feil, Robert

    2014-01-01

    Rationale Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown. Objective To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer–based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues. Methods and Results Mouse lines with smooth muscle–specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase–activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide–stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation. Conclusions These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs. PMID:23801067

  14. Derivation of Traceable and Transplantable Photoreceptors from Mouse Embryonic Stem Cells

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    Sarah Decembrini

    2014-06-01

    Full Text Available Retinal degenerative diseases resulting in the loss of photoreceptors are one of the major causes of blindness. Photoreceptor replacement therapy is a promising treatment because the transplantation of retina-derived photoreceptors can be applied now to different murine retinopathies to restore visual function. To have an unlimited source of photoreceptors, we derived a transgenic embryonic stem cell (ESC line in which the Crx-GFP transgene is expressed in photoreceptors and assessed the capacity of a 3D culture protocol to produce integration-competent photoreceptors. This culture system allows the production of a large number of photoreceptors recapitulating the in vivo development. After transplantation, integrated cells showed the typical morphology of mature rods bearing external segments and ribbon synapses. We conclude that a 3D protocol coupled with ESCs provides a safe and renewable source of photoreceptors displaying a development and transplantation competence comparable to photoreceptors from age-matched retinas.

  15. cAMP and cGMP signaling: sensory systems with prokaryotic roots adopted by eukaryotic cilia.

    Science.gov (United States)

    Johnson, Jacque-Lynne F; Leroux, Michel R

    2010-08-01

    An exciting discovery of the new millennium is that primary cilia, organelles found on most eukaryotic cells, play crucial roles in vertebrate development by modulating Hedgehog, Wnt and PDGF signaling. Analysis of the literature and sequence databases reveals that the ancient signal transduction pathway, which uses cGMP in eukaryotes or related cyclic di-GMP in bacteria, exists in virtually all eukaryotes. However, many eukaryotes that secondarily lost cilia during evolution, including flowering plants, slime molds and most fungi, lack otherwise evolutionarily conserved cGMP signaling components. Based on this intriguing phylogenetic distribution, the presence of cGMP signaling proteins within cilia, and the indispensable roles that cGMP plays in transducing environmental signals in divergent ciliated cells (e.g. vertebrate photoreceptors and Caenorhabditis elegans sensory neurons), we propose that cGMP signaling has a strong ciliary basis. cAMP signaling, also inherent to bacteria and crucial for cilium-dependent olfaction, similarly appears to have widespread usage in diverse cilia. Thus, we argue here that both cyclic nucleotides play essential and potentially ubiquitous roles in modulating ciliary functions.

  16. Photoreceptor cell dysplasia in two Tippler pigeons.

    Science.gov (United States)

    Moore, P A; Munnell, J F; Martin, C L; Prasse, K W; Carmichael, K P

    2004-01-01

    Two 12-week-old Tippler pigeons were evaluated for ocular abnormalities associated with congenital blindness. The pigeons were emaciated and blind. Biomicroscopy and direct and indirect ophthalmoscopy findings of the Tippler pigeons were normal with the exception of partially dilated pupils at rest. Scotopic (blue stimuli) and photopic monocular electroretinograms were extinguished in the blind Tippler pigeons. Histological and electron microscopy studies revealed reduced numbers of rods and cones, and an absence of the double cone complex. The photoreceptor cells' outer segments were absent, and the inner segments were short and broad. The number of cell nuclei in the outer and inner nuclear layers was decreased, and the internal and external plexiform layers were reduced in width. Photoreceptor cell endfeet with developing synaptic ribbons were present in the external plexiform layer. Inflammatory cell and subretinal debris was not seen. The electroretinographic, histopathological, and ultrastructural findings of the blind Tippler pigeons support the diagnosis of a photoreceptor cell dysplasia.

  17. Ocular anatomy and retinal photoreceptors in a skink, the sleepy lizard (Tiliqua rugosa).

    Science.gov (United States)

    New, Shaun T D; Hemmi, Jan M; Kerr, Gregory D; Bull, C Michael

    2012-10-01

    The Australian sleepy lizard (Tiliqua rugosa) is a large day-active skink which occupies stable overlapping home ranges and maintains long-term monogamous relationships. Its behavioral ecology has been extensively studied, making the sleepy lizard an ideal model for investigation of the lizard visual system and its specializations, for which relatively little is known. We examine the morphology, density, and distribution of retinal photoreceptors and describe the anatomy of the sleepy lizard eye. The sleepy lizard retina is composed solely of photoreceptors containing oil droplets, a characteristic of cones. Two groups could be distinguished; single cones and double cones, consistent with morphological descriptions of photoreceptors in other diurnal lizards. Although all photoreceptors were cone-like in morphology, a subset of photoreceptors displayed immunoreactivity to rhodopsin-the visual pigment of rods. This finding suggests that while the morphological properties of rod photoreceptors have been lost, photopigment protein composition has been conserved during evolutionary history.

  18. Dominant cone-rod dystrophy: a mouse model generated by gene targeting of the GCAP1/Guca1a gene.

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    Prateek K Buch

    Full Text Available Cone dystrophy 3 (COD3 is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1. The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG, retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions.

  19. Eyes shut homolog is required for maintaining the ciliary pocket and survival of photoreceptors in zebrafish

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    Miao Yu

    2016-11-01

    Full Text Available Mutations in the extracellular matrix protein eyes shut homolog (EYS cause photoreceptor degeneration in patients with retinitis pigmentosa 25 (RP25. Functions of EYS remain poorly understood, due in part to the lack of an EYS gene in mouse. We investigated the localization of vertebrate EYS proteins and engineered loss-of-function alleles in zebrafish. Immunostaining indicated that EYS localized near the connecting cilium/transition zone in photoreceptors. EYS also strongly localized to the cone outer segments and weakly to the rod outer segments and cone terminals in primate retinas. Analysis of mutant EYS zebrafish revealed disruption of the ciliary pocket in cone photoreceptors, indicating that EYS is required for maintaining the integrity of the ciliary pocket lumen. Mutant zebrafish exhibited progressive loss of cone and rod photoreceptors. Our results indicate that EYS protein localization is species-dependent and that EYS is required for maintaining ciliary pocket morphology and survival of photoreceptors in zebrafish.

  20. Measurement of Photon Statistics with Live Photoreceptor Cells

    CERN Document Server

    Sim, Nigel; Bessarab, Dmitri; Jones, C Michael; Krivitsky, Leonid

    2012-01-01

    We analyzed the electrophysiological response of an isolated rod photoreceptor of Xenopus laevis under stimulation by coherent and pseudo-thermal light sources. Using the suction electrode technique for single cell recordings and a fiber optics setup for light delivery allowed measurements of the major statistical characteristics of the rod response. The results indicate differences in average responses of rod cells to coherent and pseudo-thermal light of the same intensity and also differences in signal-to-noise ratios and second order intensity correlation functions. These findings should be relevant for interdisciplinary studies in applications of quantum optics in biology.

  1. Retinal photoreceptor fine structure in the red-backed salamander (Plethodon cinereus).

    Science.gov (United States)

    Braekevelt, C R

    1992-07-01

    The retinal photoreceptors of the red-backed salamander (Plethodon cinerus) have been studied by light and electron microscopy. Rods and single cones are present in this duplex retina in a ratio of about 25:1. The photoreceptors in this amphibian species are much larger than is reported for most vertebrates. In the light-adapted state, rods reach deep into the retinal epithelial (RPE) layer. The rod outer segment is composed of discs of uniform diameter displaying several very deep incisors. The rod inner segment displays a distal elliposid of mitochondria and a short stout myoid region. Rod nuclei are electron dense and often protrude through the external limiting membrane. Rod synaptic spherules are large and display several invaginated synaptic sites as well as superficial synapses. It is felt that the rods do not undergo retinomotor movements. The cone photoreceptors are much smaller than the rods and display a tapering outer segment, an unusual modified ellipsoid and a large parabolid of glycogen in the inner segment. Cone nuclei are less electron dense than rods and are located at all levels within the outer nuclear layer. The synaptic pedicle of the cones is larger, more electron lucent and display more synaptic sites (both invaginated and superficial) than that of rods. It is felt that cone photomechanical responses are minimal.

  2. Presynaptic [Ca2+] and GCAPs: aspects on the structure and function of photoreceptor ribbon synapses

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    Frank eSchmitz

    2014-02-01

    Full Text Available Changes in intracellular calcium ions [Ca2+] play important roles in photoreceptor signalling. Consequently, intracellular [Ca2+] levels need to be tightly controlled. In the light-sensitive outer segments (OS of photoreceptors, Ca2+ regulates the activity of retinal guanylate cyclases (ret-GCs thus playing a central role in phototransduction and light-adaptation by restoring light-induced decreases in cGMP. In the synaptic terminals, changes of intracellular Ca2+ trigger various aspects of neurotransmission. Photoreceptors employ tonically active ribbon synapses that encode light-induced, graded changes of membrane potential into different rates of synaptic vesicle exocytosis. The active zones of ribbon synapses contain large electron-dense structures, synaptic ribbons, that are associated with large numbers of synaptic vesicles. Synaptic coding at ribbon synapses differs from synaptic coding at conventional (phasic synapses. Recent studies revealed new insights how synaptic ribbons are involved in this process. This review focuses on the regulation of [Ca2+] in presynaptic photoreceptor terminals and on the function of a particular Ca2+-regulated protein, the neuronal calcium sensor protein GCAP2 (guanylate cyclase-activating protein-2 in the photoreceptor ribbon synapse. GCAP2, an EF hand-containing protein plays multiple roles in the OS and in the photoreceptor synapse. In the OS, GCAP2 works as a Ca2+-sensor within a Ca2+-regulated feedback loop that adjusts cGMP levels. In the photoreceptor synapse, GCAP2 binds to RIBEYE, a component of synaptic ribbons, and mediates Ca2+-dependent plasticity at that site. Possible mechanisms are discussed.

  3. Migration, integration and maturation of photoreceptor precursors following transplantation in the mouse retina.

    Science.gov (United States)

    Warre-Cornish, Katherine; Barber, Amanda C; Sowden, Jane C; Ali, Robin R; Pearson, Rachael A

    2014-05-01

    Retinal degeneration leading to loss of photoreceptors is a major cause of untreatable blindness. Recent research has yielded definitive evidence for restoration of vision following the transplantation of rod photoreceptors in murine models of blindness, while advances in stem cell biology have enabled the generation of transplantable photoreceptors from embryonic stem cells. Importantly, the amount of visual function restored is dependent upon the number of photoreceptors that migrate correctly into the recipient retina. The developmental stage of the donor cells is important for their ability to migrate; they must be immature photoreceptor precursors. Little is known about how and when donor cell migration, integration, and maturation occurs. Here, we have performed a comprehensive histological analysis of the 6-week period following rod transplantation in mice. Donor cells migrate predominately as single entities during the first week undergoing a stereotyped sequence of morphological changes in their translocation from the site of transplantation, through the interphotoreceptor matrix and into the recipient retina. This includes initial polarization toward the outer nuclear layer (ONL), followed by formation of an apical attachment and rudimentary segment during migration into the ONL. Strikingly, acquisition of a nuclear architecture typical of mature rods was accelerated compared with normal development and a feature of migrating cells. Once within the ONL, precursors formed synaptic-like structures and outer segments in accordance with normal maturation. The restoration of visual function mediated by transplanted photoreceptors correlated with the later expression of rod α-transducin, achieving maximal function by 5 weeks.

  4. Retinoic acid regulates the expression of photoreceptor transcription factor NRL.

    Science.gov (United States)

    Khanna, Hemant; Akimoto, Masayuki; Siffroi-Fernandez, Sandrine; Friedman, James S; Hicks, David; Swaroop, Anand

    2006-09-15

    NRL (neural retina leucine zipper) is a key basic motif-leucine zipper (bZIP) transcription factor, which orchestrates rod photoreceptor differentiation by activating the expression of rod-specific genes. The deletion of Nrl in mice results in functional cones that are derived from rod precursors. However, signaling pathways modulating the expression or activity of NRL have not been elucidated. Here, we show that retinoic acid (RA), a diffusible factor implicated in rod development, activates the expression of NRL in serum-deprived Y79 human retinoblastoma cells and in primary cultures of rat and porcine photoreceptors. The effect of RA is mimicked by TTNPB, a RA receptor agonist, and requires new protein synthesis. DNaseI footprinting and electrophoretic mobility shift assays (EMSA) using bovine retinal nuclear extract demonstrate that RA response elements (RAREs) identified within the Nrl promoter bind to RA receptors. Furthermore, in transiently transfected Y79 and HEK293 cells the activity of Nrl-promoter driving a luciferase reporter gene is induced by RA, and this activation is mediated by RAREs. Our data suggest that signaling by RA via RA receptors regulates the expression of NRL, providing a framework for delineating early steps in photoreceptor cell fate determination.

  5. Light adaptation and the evolution of vertebrate photoreceptors.

    Science.gov (United States)

    Morshedian, Ala; Fain, Gordon L

    2017-07-15

    Lamprey are cyclostomes, a group of vertebrates that diverged from lines leading to jawed vertebrates (including mammals) in the late Cambrian, 500 million years ago. It may therefore be possible to infer properties of photoreceptors in early vertebrate progenitors by comparing lamprey to other vertebrates. We show that lamprey rods and cones respond to light much like rods and cones in amphibians and mammals. They operate over a similar range of light intensities and adapt to backgrounds and bleaches nearly identically. These correspondences are pervasive and detailed; they argue for the presence of rods and cones very early in the evolution of vertebrates with properties much like those of rods and cones in existing vertebrate species. The earliest vertebrates were agnathans - fish-like organisms without jaws, which first appeared near the end of the Cambrian radiation. One group of agnathans became cyclostomes, which include lamprey and hagfish. Other agnathans gave rise to jawed vertebrates or gnathostomes, the group including all other existing vertebrate species. Because cyclostomes diverged from other vertebrates 500 million years ago, it may be possible to infer some of the properties of the retina of early vertebrate progenitors by comparing lamprey to other vertebrates. We have previously shown that rods and cones in lamprey respond to light much like photoreceptors in other vertebrates and have a similar sensitivity. We now show that these affinities are even closer. Both rods and cones adapt to background light and to bleaches in a manner almost identical to other vertebrate photoreceptors. The operating range in darkness is nearly the same in lamprey and in amphibian or mammalian rods and cones; moreover background light shifts response-intensity curves downward and to the right over a similar range of ambient intensities. Rods show increment saturation at about the same intensity as mammalian rods, and cones never saturate. Bleaches decrease

  6. Specialized photoreceptor composition in the raptor fovea.

    Science.gov (United States)

    Mitkus, Mindaugas; Olsson, Peter; Toomey, Matthew B; Corbo, Joseph C; Kelber, Almut

    2017-02-15

    The retinae of many bird species contain a depression with high photoreceptor density known as the fovea. Many species of raptors have two foveae, a deep central fovea and a shallower temporal fovea. Birds have six types of photoreceptors: rods, active in dim light, double cones that are thought to mediate achromatic discrimination, and four types of single cones mediating color vision. To maximize visual acuity, the fovea should only contain photoreceptors contributing to high-resolution vision. Interestingly, it has been suggested that raptors might lack double cones in the fovea. We used transmission electron microscopy and immunohistochemistry to evaluate this claim in five raptor species: the common buzzard (Buteo buteo), the honey buzzard (Pernis apivorus), the Eurasian sparrowhawk (Accipiter nisus), the red kite (Milvus milvus) and the peregrine falcon (Falco peregrinus). We found that all species, except the Eurasian sparrowhawk, lack double cones in the center of the central fovea. The size of the double cone-free zone differed between species. Only the common buzzard had a double cone-free zone in the temporal fovea. In three species, we examined opsin expression in the central fovea and found evidence that rod opsin positive cells were absent and violet-sensitive cone and green-sensitive cone opsin positive cells were present. We conclude that not only double cones, but also single cones may contribute to high-resolution vision in birds, and that raptors may in fact possess high-resolution tetrachromatic vision in the central fovea. This article is protected by copyright. All rights reserved.

  7. Distinct and atypical intrinsic and extrinsic cell death pathways between photoreceptor cell types upon specific ablation of Ranbp2 in cone photoreceptors.

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    Kyoung-In Cho

    2013-06-01

    Full Text Available Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2, a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+ -apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct

  8. Cone rod dystrophies

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    Hamel Christian P

    2007-02-01

    Full Text Available Abstract Cone rod dystrophies (CRDs (prevalence 1/40,000 are inherited retinal dystrophies that belong to the group of pigmentary retinopathies. CRDs are characterized by retinal pigment deposits visible on fundus examination, predominantly localized to the macular region. In contrast to typical retinitis pigmentosa (RP, also called the rod cone dystrophies (RCDs resulting from the primary loss in rod photoreceptors and later followed by the secondary loss in cone photoreceptors, CRDs reflect the opposite sequence of events. CRD is characterized by primary cone involvement, or, sometimes, by concomitant loss of both cones and rods that explains the predominant symptoms of CRDs: decreased visual acuity, color vision defects, photoaversion and decreased sensitivity in the central visual field, later followed by progressive loss in peripheral vision and night blindness. The clinical course of CRDs is generally more severe and rapid than that of RCDs, leading to earlier legal blindness and disability. At end stage, however, CRDs do not differ from RCDs. CRDs are most frequently non syndromic, but they may also be part of several syndromes, such as Bardet Biedl syndrome and Spinocerebellar Ataxia Type 7 (SCA7. Non syndromic CRDs are genetically heterogeneous (ten cloned genes and three loci have been identified so far. The four major causative genes involved in the pathogenesis of CRDs are ABCA4 (which causes Stargardt disease and also 30 to 60% of autosomal recessive CRDs, CRX and GUCY2D (which are responsible for many reported cases of autosomal dominant CRDs, and RPGR (which causes about 2/3 of X-linked RP and also an undetermined percentage of X-linked CRDs. It is likely that highly deleterious mutations in genes that otherwise cause RP or macular dystrophy may also lead to CRDs. The diagnosis of CRDs is based on clinical history, fundus examination and electroretinogram. Molecular diagnosis can be made for some genes, genetic counseling is

  9. Pineal photoreceptor cells are required for maintaining the circadian rhythms of behavioral visual sensitivity in zebrafish.

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    Xinle Li

    Full Text Available In non-mammalian vertebrates, the pineal gland functions as the central pacemaker that regulates the circadian rhythms of animal behavior and physiology. We generated a transgenic zebrafish line [Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry] in which the E. coli nitroreductase is expressed in pineal photoreceptor cells. In developing embryos and young adults, the transgene is expressed in both retinal and pineal photoreceptor cells. During aging, the expression of the transgene in retinal photoreceptor cells gradually diminishes. By 8 months of age, the Gnat2 promoter-driven nitroreductase is no longer expressed in retinal photoreceptor cells, but its expression in pineal photoreceptor cells persists. This provides a tool for selective ablation of pineal photoreceptor cells, i.e., by treatments with metronidazole. In the absence of pineal photoreceptor cells, the behavioral visual sensitivity of the fish remains unchanged; however, the circadian rhythms of rod and cone sensitivity are diminished. Brief light exposures restore the circadian rhythms of behavioral visual sensitivity. Together, the data suggest that retinal photoreceptor cells respond to environmental cues and are capable of entraining the circadian rhythms of visual sensitivity; however, they are insufficient for maintaining the rhythms. Cellular signals from the pineal photoreceptor cells may be required for maintaining the circadian rhythms of visual sensitivity.

  10. Control of guanylate cyclase activity in the rod outer segment.

    Science.gov (United States)

    Pannbacker, R G

    1973-12-14

    Mammalian photoreceptors contain a guanylate cyclase which has a high specific activity and is inhibited by exposure of the rod outer segment to light. Several minutes are required for this inhibition to take effect, indicating that it is not a step in visual excitation. The activity of the enzyme is sensitive to the concentration of calcium ion in the medium, suggesting that light-induced changes in calcium distribution in the photoreceptor could control guanylate cyclase activity.

  11. Special characteristics of the transcription and splicing machinery in photoreceptor cells of the mammalian retina.

    Science.gov (United States)

    Derlig, Kristin; Giessl, Andreas; Brandstätter, Johann Helmut; Enz, Ralf; Dahlhaus, Regina

    2015-11-01

    Chromatin organization and the management of transcription and splicing are fundamental to the correct functioning of every cell but, in particular, for highly active cells such as photoreceptors, the sensory neurons of the retina. Rod photoreceptor cells of nocturnal animals have recently been shown to have an inverted chromatin architecture compared with rod photoreceptor cells of diurnal animals. The heterochromatin is concentrated in the center of the nucleus, whereas the genetically active euchromatin is positioned close to the nuclear membrane. This unique chromatin architecture suggests that the transcription and splicing machinery is also subject to specific adaptations in these cells. Recently, we described the protein Simiate, which is enriched in nuclear speckles and seems to be involved in transcription and splicing processes. Here, we examine the distribution of Simiate and nuclear speckles in neurons of mouse retinae. In retinal neurons of the inner nuclear and ganglion cell layer, Simiate is concentrated in a clustered pattern in the nuclear interior, whereas in rod and cone photoreceptor cells, Simiate is present at the nuclear periphery. Further staining with markers for the transcription and splicing machinery has confirmed the localization of nuclear speckle components at the periphery. Comparing the distribution of nuclear speckles in retinae of the nocturnal mouse with the diurnal degu, we found no differences in the arrangement of the transcription and splicing machinery in their photoreceptor cells, thus suggesting that the organization of these machineries is not related to the animal's lifestyle but rather represents a general characteristic of photoreceptor organization and function.

  12. Integrity of the cone photoreceptor mosaic in oligocone trichromacy

    DEFF Research Database (Denmark)

    Michaelides, Michel; Rha, Jungtae; Dees, Elise W;

    2011-01-01

    Oligocone trichromacy (OT) is an unusual cone dysfunction syndrome characterized by reduced visual acuity, mild photophobia, reduced amplitude of the cone electroretinogram with normal rod responses, normal fundus appearance, and normal or near-normal color vision. It has been proposed that these...... that these patients have a reduced number of normal functioning cones (oligocone). This paper has sought to evaluate the integrity of the cone photoreceptor mosaic in four patients previously described as having OT....

  13. Rod and Rod-driven Function in Achromatopsia and Blue Cone Monochromatism

    Science.gov (United States)

    Moskowitz, Anne; Hansen, Ronald M.; Akula, James D.; Eklund, Susan E.; Fulton, Anne B.

    2008-01-01

    Purpose To evaluate rod photoreceptor and postreceptor retinal function in pediatric patients with achromatopsia (ACHR) and blue cone monochromatism (BCM) using contemporary electroretinographic (ERG) procedures. Methods Fifteen patients (age 1 to 20 years) with ACHR and six patients (age 4 to 22 years) with BCM were studied. ERG responses to full-field stimuli were obtained in scotopic and photopic conditions. Rod photoreceptor (Srod, Rrod) and rod-driven postreceptor (log σ, Vmax) response parameters were calculated from the a-wave and b-wave. The ERG records were digitally filtered to demonstrate the oscillatory potentials (OPs); a sensitivity parameter, log SOPA1/2, and an amplitude parameter, SOPAmax, were used to characterize the OP response. Response parameters were compared to those of 12 normal control subjects. Results As expected, photopic responses were non-detectable in patients with ACHR and BCM. In addition, mean scotopic photoreceptor (Rrod) and postreceptor (Vmax and SOPAmax) amplitude parameters were significantly reduced compared to those in normal controls. The flash intensity required to evoke a half maximum b-wave amplitude (log σ) was significantly increased. Conclusions The results of this study provide evidence that deficits in rod and rod mediated function occur in the primary cone dysfunction syndromes, achromatopsia and blue cone monochromatism. PMID:18824728

  14. Identifying photoreceptors in blind eyes caused by RPE65 mutations: Prerequisite for human gene therapy success.

    Science.gov (United States)

    Jacobson, Samuel G; Aleman, Tomas S; Cideciyan, Artur V; Sumaroka, Alexander; Schwartz, Sharon B; Windsor, Elizabeth A M; Traboulsi, Elias I; Heon, Elise; Pittler, Steven J; Milam, Ann H; Maguire, Albert M; Palczewski, Krzysztof; Stone, Edwin M; Bennett, Jean

    2005-04-26

    Mutations in RPE65, a gene essential to normal operation of the visual (retinoid) cycle, cause the childhood blindness known as Leber congenital amaurosis (LCA). Retinal gene therapy restores vision to blind canine and murine models of LCA. Gene therapy in blind humans with LCA from RPE65 mutations may also have potential for success but only if the retinal photoreceptor layer is intact, as in the early-disease stage-treated animals. Here, we use high-resolution in vivo microscopy to quantify photoreceptor layer thickness in the human disease to define the relationship of retinal structure to vision and determine the potential for gene therapy success. The normally cone photoreceptor-rich central retina and rod-rich regions were studied. Despite severely reduced cone vision, many RPE65-mutant retinas had near-normal central microstructure. Absent rod vision was associated with a detectable but thinned photoreceptor layer. We asked whether abnormally thinned RPE65-mutant retina with photoreceptor loss would respond to treatment. Gene therapy in Rpe65(-/-) mice at advanced-disease stages, a more faithful mimic of the humans we studied, showed success but only in animals with better-preserved photoreceptor structure. The results indicate that identifying and then targeting retinal locations with retained photoreceptors will be a prerequisite for successful gene therapy in humans with RPE65 mutations and in other retinal degenerative disorders now moving from proof-of-concept studies toward clinical trials.

  15. Acute Zonal Cone Photoreceptor Outer Segment Loss.

    Science.gov (United States)

    Aleman, Tomas S; Sandhu, Harpal S; Serrano, Leona W; Traband, Anastasia; Lau, Marisa K; Adamus, Grazyna; Avery, Robert A

    2017-05-01

    The diagnostic path presented narrows down the cause of acute vision loss to the cone photoreceptor outer segment and will refocus the search for the cause of similar currently idiopathic conditions. To describe the structural and functional associations found in a patient with acute zonal occult photoreceptor loss. A case report of an adolescent boy with acute visual field loss despite a normal fundus examination performed at a university teaching hospital. Results of a complete ophthalmic examination, full-field flash electroretinography (ERG) and multifocal ERG, light-adapted achromatic and 2-color dark-adapted perimetry, and microperimetry. Imaging was performed with spectral-domain optical coherence tomography (SD-OCT), near-infrared (NIR) and short-wavelength (SW) fundus autofluorescence (FAF), and NIR reflectance (REF). The patient was evaluated within a week of the onset of a scotoma in the nasal field of his left eye. Visual acuity was 20/20 OU, and color vision was normal in both eyes. Results of the fundus examination and of SW-FAF and NIR-FAF imaging were normal in both eyes, whereas NIR-REF imaging showed a region of hyporeflectance temporal to the fovea that corresponded with a dense relative scotoma noted on light-adapted static perimetry in the left eye. Loss in the photoreceptor outer segment detected by SD-OCT co-localized with an area of dense cone dysfunction detected on light-adapted perimetry and multifocal ERG but with near-normal rod-mediated vision according to results of 2-color dark-adapted perimetry. Full-field flash ERG findings were normal in both eyes. The outer nuclear layer and inner retinal thicknesses were normal. Localized, isolated cone dysfunction may represent the earliest photoreceptor abnormality or a distinct entity within the acute zonal occult outer retinopathy complex. Acute zonal occult outer retinopathy should be considered in patients with acute vision loss and abnormalities on NIR-REF imaging, especially if

  16. Ciliary photoreceptors in the cerebral eyes of a protostome larva

    Directory of Open Access Journals (Sweden)

    Passamaneck Yale J

    2011-03-01

    Full Text Available Abstract Background Eyes in bilaterian metazoans have been described as being composed of either ciliary or rhabdomeric photoreceptors. Phylogenetic distribution, as well as distinct morphologies and characteristic deployment of different photopigments (ciliary vs. rhabdomeric opsins and transduction pathways argue for the co-existence of both of these two photoreceptor types in the last common bilaterian ancestor. Both receptor types exist throughout the Bilateria, but only vertebrates are thought to use ciliary photoreceptors for directional light detection in cerebral eyes, while all other invertebrate bilaterians studied utilize rhabdomeric photoreceptors for this purpose. In protostomes, ciliary photoreceptors that express c-opsin have been described only from a non-visual deep-brain photoreceptor. Their homology with vertebrate rods and cones of the human eye has been hypothesized to represent a unique functional transition from non-visual to visual roles in the vertebrate lineage. Results To test the hypothesis that protostome cerebral eyes employ exclusively rhabdomeric photoreceptors, we investigated the ultrastructure of the larval eyes in the brachiopod Terebratalia transversa. We show that these pigment-cup eyes consist of a lens cell and a shading pigment cell, both of which are putative photoreceptors, deploying a modified, enlarged cilium for light perception, and have axonal connections to the larval brain. Our investigation of the gene expression patterns of c-opsin, Pax6 and otx in these eyes confirms that the larval eye spots of brachiopods are cerebral eyes that deploy ciliary type photoreceptors for directional light detection. Interestingly, c-opsin is also expressed during early embryogenesis in all potential apical neural cells, becoming restricted to the anterior neuroectoderm, before expression is initiated in the photoreceptor cells of the eyes. Coincident with the expression of c-opsin in the presumptive neuroectoderm

  17. Overlap of abnormal photoreceptor development and progressive degeneration in Leber congenital amaurosis caused by NPHP5 mutation.

    Science.gov (United States)

    Downs, Louise M; Scott, Erin M; Cideciyan, Artur V; Iwabe, Simone; Dufour, Valerie; Gardiner, Kristin L; Genini, Sem; Marinho, Luis Felipe; Sumaroka, Alexander; Kosyk, Mychajlo S; Swider, Malgorzata; Aguirre, Geoffrey K; Jacobson, Samuel G; Beltran, William A; Aguirre, Gustavo D

    2016-10-01

    Ciliary defects can result in severe disorders called ciliopathies. Mutations in NPHP5 cause a ciliopathy characterized by severe childhood onset retinal blindness, Leber congenital amaurosis (LCA), and renal disease. Using the canine NPHP5-LCA model we compared human and canine retinal phenotypes, and examined the early stages of photoreceptor development and degeneration, the kinetics of photoreceptor loss, the progression of degeneration and the expression profiles of selected genes. NPHP5-mutant dogs recapitulate the human phenotype of very early loss of rods, and relative retention of the central retinal cone photoreceptors that lack function. In mutant dogs, rod and cone photoreceptors have a sensory cilium, but develop and function abnormally and then rapidly degenerate; L/M cones are more severely affected than S-cones. The lack of outer segments in mutant cones indicates a ciliary dysfunction. Genes expressed in mutant rod or both rod and cone photoreceptors show significant downregulation, while those expressed only in cones are unchanged. Many genes in cell-death and -survival pathways also are downregulated. The canine disease is a non-syndromic LCA-ciliopathy, with normal renal structures and no CNS abnormalities. Our results identify the critical time points in the pathogenesis of the photoreceptor disease, and bring us closer to defining a potential time window for testing novel therapies for translation to patients.

  18. Comparative investigation of stimulus-evoked rod outer segment movement and retinal electrophysiological activity

    Science.gov (United States)

    Lu, Yiming; Wang, Benquan; Yao, Xincheng

    2017-02-01

    Transient retinal phototropism (TRP) has been observed in rod photoreceptors activated by oblique visible light flashes. Time-lapse confocal microscopy and optical coherence tomography (OCT) revealed rod outer segment (ROS) movements as the physical source of TRP. However, the physiological source of TRP is still not well understood. In this study, concurrent TRP and electroretinogram (ERG) measurements disclosed a remarkably earlier onset time of the ROS movements (low sodium treatment reversibly blocked the photoreceptor ERG a-wave, which is known to reflect hyperpolarization of retinal photoreceptors, but preserved the TRP associated rod OS movements well. Our experimental results and theoretical analysis suggested that the physiological source of TRP might be attributed to early stages of phototransduction, before the hyperpolarization of retinal photoreceptors.

  19. cGMP signalling : different ways to create a pathway

    NARCIS (Netherlands)

    Roelofs, Jeroen; Smith, Janet L.; Haastert, Peter J.M. van

    2003-01-01

    Recently, a novel cGMP signalling cascade was uncovered in Dictyostelium, a eukaryote that diverged from the lineage leading to metazoa after plants and before yeast. In both Dictyostelium and metazoa, the ancient cAMP-binding (cNB) motif of bacterial CAP has been modified and assembled with other d

  20. Morphoelastic rods

    CERN Document Server

    Tiero, Alessandro

    2014-01-01

    We propose a mechanical theory describing elastic rods which, like plant organs, can grow and can change their intrinsic curvature and torsion. The equations ruling accretion and remodeling are obtained by combining balance laws involving non-standard forces with constitutive prescriptions filtered by a dissipation principle that takes into account both standard and non-standard working.

  1. Protective gene expression changes elicited by an inherited defect in photoreceptor structure.

    Directory of Open Access Journals (Sweden)

    Yagya V Sharma

    Full Text Available Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE, and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model.

  2. Functional and Molecular Characterization of Rod-like Cells from Retinal Stem Cells Derived from the Adult Ciliary Epithelium

    OpenAIRE

    Gian Carlo Demontis; Claudia Aruta; Antonella Comitato; Anna De Marzo; Valeria Marigo

    2012-01-01

    In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor...

  3. CONTROL ROD

    Science.gov (United States)

    Zinn, W.H.; Ross, H.V.

    1958-11-18

    A control rod is described for a nuclear reactor. In certaln reactor designs it becomes desirable to use a control rod having great width but relatively llttle thickness. This patent is addressed to such a need. The neutron absorbing material is inserted in a triangular tube, leaving volds between the circular insert and the corners of the triangular tube. The material is positioned within the tube by the use of dummy spacers to achleve the desired absorption pattern, then the ends of the tubes are sealed with suitable plugs. The tubes may be welded or soldered together to form two flat surfaces of any desired width, and covered with sheetmetal to protect the tubes from damage. This design provides a control member that will not distort under the action of outside forces or be ruptured by gases generated within the jacketed control member.

  4. The transcription factor neural retina leucine zipper (NRL) controls photoreceptor-specific expression of myocyte enhancer factor Mef2c from an alternative promoter.

    Science.gov (United States)

    Hao, Hong; Tummala, Padmaja; Guzman, Eduardo; Mali, Raghuveer S; Gregorski, Janina; Swaroop, Anand; Mitton, Kenneth P

    2011-10-07

    Neural retina leucine zipper (NRL) is an essential transcription factor for cell fate specification and functional maintenance of rod photoreceptors in the mammalian retina. In the Nrl(-/-) mouse retina, photoreceptor precursors fail to produce rods and generate functional cone photoreceptors that predominantly express S-opsin. Previous global expression analysis using microarrays revealed dramatically reduced expression of myocyte enhancer factor Mef2c in the adult Nrl(-/-) retina. We undertook this study to examine the biological relevance of Mef2c expression in retinal rod photoreceptors. Bioinformatics analysis, rapid analysis of cDNA ends (5'-RACE), and reverse transcription coupled with qPCR using splice site-specific oligonucleotides suggested that Mef2c is expressed in the mature retina from an alternative promoter. Chromatin immunoprecipitation (ChIP) studies showed the association of active RNA polymerase II and acetylated histone H3 just upstream of Mef2c exon 4, providing additional evidence for the utilization of an alternative promoter in the retina. In concordance, we observed the binding of NRL to a putative NRL-response element (NRE) at this location by ChIP-seq and electrophoretic mobility shift assays. NRL also activated the Mef2c alternative promoter in vitro and in vivo. Notably, MEF2C could support Rhodopsin promoter activity in rod photoreceptors. We conclude that Mef2c expression from an alternative promoter in the retina is regulated by NRL. Our studies also implicate MEF2C as a transcriptional regulator of homeostasis in rod photoreceptor cells.

  5. Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse.

    Science.gov (United States)

    Daniele, Lauren L; Lillo, Concepcion; Lyubarsky, Arkady L; Nikonov, Sergei S; Philp, Nancy; Mears, Alan J; Swaroop, Anand; Williams, David S; Pugh, Edward N

    2005-06-01

    To test the hypothesis that Nrl(-)(/)(-) photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. The photoreceptor layer of fixed retinal tissue of 4- to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG. Outer segments of Nrl(-/-) photoreceptors ( approximately 7 mum) are shorter than those of wild-type (WT) rods ( approximately 25 mum) and cones ( approximately 15 mum); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl(-/-) photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl(-/-) a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the lambda(max) of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was approximately 50 ms, more than two times faster than that of rods. Many morphological, molecular, and electrophysiological features of the Nrl(-/-) photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.

  6. Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.

    Science.gov (United States)

    Beltran, William A; Cideciyan, Artur V; Lewin, Alfred S; Iwabe, Simone; Khanna, Hemant; Sumaroka, Alexander; Chiodo, Vince A; Fajardo, Diego S; Román, Alejandro J; Deng, Wen-Tao; Swider, Malgorzata; Alemán, Tomas S; Boye, Sanford L; Genini, Sem; Swaroop, Anand; Hauswirth, William W; Jacobson, Samuel G; Aguirre, Gustavo D

    2012-02-07

    Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.

  7. Detection of single photons by toad and mouse rods.

    Science.gov (United States)

    Reingruber, Jürgen; Pahlberg, Johan; Woodruff, Michael L; Sampath, Alapakkam P; Fain, Gordon L; Holcman, David

    2013-11-26

    Amphibian and mammalian rods can both detect single photons of light even though they differ greatly in physical dimensions, mammalian rods being much smaller in diameter than amphibian rods. To understand the changes in physiology and biochemistry required by such large differences in outer segment geometry, we developed a computational approach, taking into account the spatial organization of the outer segment divided into compartments, together with molecular dynamics simulations of the signaling cascade. We generated simulations of the single-photon response together with intrinsic background fluctuations in toad and mouse rods. Combining this computational approach with electrophysiological data from mouse rods, we determined key biochemical parameters. On average around one phosphodiesterase (PDE) molecule is spontaneously active per mouse compartment, similar to the value for toad, which is unexpected due to the much smaller diameter in mouse. A larger number of spontaneously active PDEs decreases dark noise, thereby improving detection of single photons; it also increases cGMP turnover, which accelerates the decay of the light response. These constraints explain the higher PDE density in mammalian compared with amphibian rods that compensates for the much smaller diameter of mammalian disks. We further find that the rate of cGMP hydrolysis by light-activated PDE is diffusion limited, which is not the case for spontaneously activated PDE. As a consequence, in the small outer segment of a mouse rod only a few activated PDEs are sufficient to generate a signal that overcomes noise, which permits a shorter lifetime of activated rhodopsin and greater temporal resolution.

  8. Bifurcation analysis of a photoreceptor interaction model for Retinitis Pigmentosa

    Science.gov (United States)

    Camacho, Erika T.; Radulescu, Anca; Wirkus, Stephen

    2016-09-01

    Retinitis Pigmentosa (RP) is the term used to describe a diverse set of degenerative eye diseases affecting the photoreceptors (rods and cones) in the retina. This work builds on an existing mathematical model of RP that focused on the interaction of the rods and cones. We non-dimensionalize the model and examine the stability of the equilibria. We then numerically investigate other stable modes that are present in the system for various parameter values and relate these modes to the original problem. Our results show that stable modes exist for a wider range of parameter values than the stability of the equilibrium solutions alone, suggesting that additional approaches to preventing cone death may exist.

  9. A role for prenylated rab acceptor 1 in vertebrate photoreceptor development

    Directory of Open Access Journals (Sweden)

    Dickison Virginia M

    2012-12-01

    Full Text Available Abstract Background The rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day (P 10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration. Results Using a microarray approach, we performed gene profiling comparing rd1 and wild type (wt retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1, which codes for the protein Prenylated rab acceptor 1 (PRA1 and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking. Conclusions We have identified genes that are differentially regulated in the rd1 retina at early

  10. Bipolar Cell-Photoreceptor Connectivity in the Zebrafish (Danio rerio) Retina

    Science.gov (United States)

    Li, Yong N.; Tsujimura, Taro; Kawamura, Shoji; Dowling, John E.

    2013-01-01

    Bipolar cells convey luminance, spatial and color information from photoreceptors to amacrine and ganglion cells. We studied the photoreceptor connectivity of 321 bipolar cells in the adult zebrafish retina. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was inserted into whole-mounted transgenic zebrafish retinas to label bipolar cells. The photoreceptors that connect to these DiI-labeled cells were identified by transgenic fluorescence or their positions relative to the fluorescent cones, as cones are arranged in a highly-ordered mosaic: rows of alternating blue- (B) and ultraviolet-sensitive (UV) single cones alternate with rows of red- (R) and green-sensitive (G) double cones. Rod terminals intersperse among cone terminals. As many as 18 connectivity subtypes were observed, 9 of which – G, GBUV, RG, RGB, RGBUV, RGRod, RGBRod, RGBUVRod and RRod bipolar cells – accounted for 96% of the population. Based on their axon terminal stratification, these bipolar cells could be further sub-divided into ON, OFF, and ON-OFF cells. The dendritic spread size, soma depth and size, and photoreceptor connections of the 308 bipolar cells within the 9 common connectivity subtypes were determined, and their dendritic tree morphologies and axonal stratification patterns compared. We found that bipolar cells with the same axonal stratification patterns could have heterogeneous photoreceptor connectivity whereas bipolar cells with the same dendritic tree morphology usually had the same photoreceptor connectivity, although their axons might stratify on different levels. PMID:22907678

  11. Midkine-a protein localization in the developing and adult retina of the zebrafish and its function during photoreceptor regeneration.

    Directory of Open Access Journals (Sweden)

    Esther Gramage

    Full Text Available Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod

  12. Requirement of histone deacetylase activity for the expression of critical photoreceptor genes

    Directory of Open Access Journals (Sweden)

    Cepko Constance L

    2007-06-01

    Full Text Available Abstract Background Histone deacetylases (HDACs play a major role in the regulation of gene transcription, often leading to transcriptional repression, as well as other effects following deacetylation of non-histone proteins. Results To investigate the role of HDACs in the developing mammalian retina, a general inhibitor of HDACs, trichostatin-A (TSA, was used to treat newborn murine retinae in explant cultures. Inhibition of HDAC activity resulted in a reduction in RNA levels for genes that regulate retinal development, as well as cell cycle regulators. Several of the genes encode transcription factors essential for rod photoreceptor development, Otx2, Nrl, and Crx. Using luciferase reporter assays, the promoter activity of both Nrl and Crx was found to be compromised by HDAC inhibition. Furthermore, downregulation of gene expression by HDAC inhibition didn't require de novo protein synthesis, and was associated with hyperacetylation of histones and non-histone proteins. Finally, HDAC inhibition in retinal explant cultures resulted in increased cell death, reduction in proliferation, a complete loss of rod photoreceptors and Müller glial cells, and an increase in bipolar cells. Conclusion HDAC activity is required for the expression of critical pro-rod transcription factors and the development of rod photoreceptor cells.

  13. Calcium channel-dependent molecular maturation of photoreceptor synapses.

    Directory of Open Access Journals (Sweden)

    Nawal Zabouri

    Full Text Available Several studies have shown the importance of calcium channels in the development and/or maturation of synapses. The Ca(V1.4(α(1F knockout mouse is a unique model to study the role of calcium channels in photoreceptor synapse formation. It features abnormal ribbon synapses and aberrant cone morphology. We investigated the expression and targeting of several key elements of ribbon synapses and analyzed the cone morphology in the Ca(V1.4(α(1F knockout retina. Our data demonstrate that most abnormalities occur after eye opening. Indeed, scaffolding proteins such as Bassoon and RIM2 are properly targeted at first, but their expression and localization are not maintained in adulthood. This indicates that either calcium or the Ca(V1.4 channel, or both are necessary for the maintenance of their normal expression and distribution in photoreceptors. Other proteins, such as Veli3 and PSD-95, also display abnormal expression in rods prior to eye opening. Conversely, vesicle related proteins appear normal. Our data demonstrate that the Ca(V1.4 channel is important for maintaining scaffolding proteins in the ribbon synapse but less vital for proteins related to vesicular release. This study also confirms that in adult retinae, cones show developmental features such as sprouting and synaptogenesis. Overall we present evidence that in the absence of the Ca(V1.4 channel, photoreceptor synapses remain immature and are unable to stabilize.

  14. Regulation of cGMP synthesis in cultured podocytes by vasoactive hormones.

    Science.gov (United States)

    Lewko, B; Gołos, M; Latawiec, E; Angielski, S; Stepinski, J

    2006-12-01

    The podocytes are highly differentiated cells playing a key role in glomerular filtration. Vasoactive factors including angiotensin II (Ang II) and cyclic guanosine 5' monophosphate (cGMP) are synthesized by these cells upon stimulation as well as in the basal state. In this study we have tested whether angiotensin II affects the total synthesis of cGMP in primary culture of rat podocytes. The cells were stimulated with atrial natriuretic peptide (ANP) and/or a nitric oxide (NO) donor, S-nitroso-N-acetyl penicillamine (SNAP), in the absence or presence of Ang II. The cGMP synthesis was determined by radioimmunoassay (RIA). ANP or SNAP alone increased the cGMP synthesis in podocytes although the effects were not additive unless Ang II was present in the medium. Ang II suppressed the ANP-dependent cGMP synthesis whereas SNAP-dependent cGMP production remained unaffected. These effects were prevented by a non-specific antagonist of Ang II receptors (AT), saralasin. Adversely, PD123319, a specific inhibitor of AT2 receptors, augmented inhibition of ANP-dependent and enhanced the NO-dependent cGMP production. Probenecid, an inhibitor of cGMP extrusion from the cells, suppressed the cGMP generation by both ANP and SNAP. We conclude that cGMP synthesis in cultured podocytes is modulated by angiotensin II and that two adversely acting receptors, AT1 and AT2 are involved in this effect. Additionally, production of cGMP might be intrinsically inhibited by cGMP accumulating inside the cells.

  15. Design of CGMP Production of 18F- and 68Ga-Radiopharmaceuticals

    OpenAIRE

    Yen-Ting Chi; Pei-Chun Chu; Hao-Yu Chao; Wei-Chen Shieh; Chen, Chuck C.

    2014-01-01

    Objective. Radiopharmaceutical production process must adhere to current good manufacturing process (CGMP) compliance to ensure the quality of precursor, prodrug (active pharmaceutical ingredient, API), and the final drug product that meet acceptance criteria. We aimed to develop an automated system for production of CGMP grade of PET radiopharmaceuticals. Methods. The hardware and software of the automated synthesizer that fit in the hot cell under cGMP requirement were developed. Examples...

  16. Cav1.4 L-Type Calcium Channels Contribute to Calpain Activation in Degenerating Photoreceptors of rd1 Mice.

    Directory of Open Access Journals (Sweden)

    Christian Schön

    Full Text Available Retinitis pigmentosa is an inherited blinding disorder characterized by progressive degeneration and loss of photoreceptors. The exact mechanism of degeneration and cell death of photoreceptors is not known, but is thought to involve disturbed Ca2+-signaling. Ca2+ can enter the photoreceptor cell via outer segment cyclic nucleotide-gated (CNG channels or synaptic Cav1.4 L-type voltage-gated calcium channels (VGCC. Previously, we have shown that genetic ablation of the Cngb1 gene encoding the B subunit of the rod CNG channel delays the fast progressing degeneration in the rd1 mutant mouse model of retinitis pigmentosa. In this study, we crossbred rd1 mice with the Cacna1f-deficient mouse lacking the Cav1.4 α1 subunit of the L-type VGCC. Longitudinal in vivo examinations of photoreceptor layer thickness by optical coherence tomography revealed a significant, but not sustained delay of retinal degeneration in Cacna1f x rd1 double mutant mice compared to rd1 mice. This was accompanied by a reduction of TUNEL positive cells in the early phase of rod degeneration. Remarkably, Cacna1f x rd1 double mutant mice displayed a strong decrease in the activation of the Ca2+-dependent protease calpain during photoreceptor loss. Our results show that genetic deletion of the synaptic Cav1.4 L-type VGCCs impairs calpain activation and leads to a short-term preservation of photoreceptors in the rd1 mouse.

  17. The influence of photoreceptor size and distribution on optical sensitivity in the eyes of lanternfishes (Myctophidae.

    Directory of Open Access Journals (Sweden)

    Fanny de Busserolles

    Full Text Available The mesopelagic zone of the deep-sea (200-1000 m is characterised by exponentially diminishing levels of downwelling sunlight and by the predominance of bioluminescence emissions. The ability of mesopelagic organisms to detect and behaviourally react to downwelling sunlight and/or bioluminescence will depend on the visual task and ultimately on the eyes and their capacity for detecting low levels of illumination and intermittent point sources of bioluminescent light. In this study, we investigate the diversity of the visual system of the lanternfish (Myctophidae. We focus specifically on the photoreceptor cells by examining their size, arrangement, topographic distribution and contribution to optical sensitivity in 53 different species from 18 genera. We also examine the influence(s of both phylogeny and ecology on these photoreceptor variables using phylogenetic comparative analyses in order to understand the constraints placed on the visual systems of this large group of mesopelagic fishes at the first stage of retinal processing. We report great diversity in the visual system of the Myctophidae at the level of the photoreceptors. Photoreceptor distribution reveals clear interspecific differences in visual specialisations (areas of high rod photoreceptor density, indicating potential interspecific differences in interactions with prey, predators and/or mates. A great diversity in photoreceptor design (length and diameter and density is also present. Overall, the myctophid eye is very sensitive compared to other teleosts and each species seems to be specialised for the detection of a specific signal (downwelling light or bioluminescence, potentially reflecting different visual demands for survival. Phylogenetic comparative analyses highlight several relationships between photoreceptor characteristics and the ecological variables tested (depth distribution and luminous tissue patterns. Depth distribution at night was a significant factor in most

  18. The influence of photoreceptor size and distribution on optical sensitivity in the eyes of lanternfishes (Myctophidae)

    KAUST Repository

    de Busserolles, Fanny

    2014-06-13

    The mesopelagic zone of the deep-sea (200-1000 m) is characterised by exponentially diminishing levels of downwelling sunlight and by the predominance of bioluminescence emissions. The ability of mesopelagic organisms to detect and behaviourally react to downwelling sunlight and/or bioluminescence will depend on the visual task and ultimately on the eyes and their capacity for detecting low levels of illumination and intermittent point sources of bioluminescent light. In this study, we investigate the diversity of the visual system of the lanternfish (Myctophidae). We focus specifically on the photoreceptor cells by examining their size, arrangement, topographic distribution and contribution to optical sensitivity in 53 different species from 18 genera. We also examine the influence(s) of both phylogeny and ecology on these photoreceptor variables using phylogenetic comparative analyses in order to understand the constraints placed on the visual systems of this large group of mesopelagic fishes at the first stage of retinal processing. We report great diversity in the visual system of the Myctophidae at the level of the photoreceptors. Photoreceptor distribution reveals clear interspecific differences in visual specialisations (areas of high rod photoreceptor density), indicating potential interspecific differences in interactions with prey, predators and/or mates. A great diversity in photoreceptor design (length and diameter) and density is also present. Overall, the myctophid eye is very sensitive compared to other teleosts and each species seems to be specialised for the detection of a specific signal (downwelling light or bioluminescence), potentially reflecting different visual demands for survival. Phylogenetic comparative analyses highlight several relationships between photoreceptor characteristics and the ecological variables tested (depth distribution and luminous tissue patterns). Depth distribution at night was a significant factor in most of the

  19. Bat eyes have ultraviolet-sensitive cone photoreceptors.

    Directory of Open Access Journals (Sweden)

    Brigitte Müller

    Full Text Available Mammalian retinae have rod photoreceptors for night vision and cone photoreceptors for daylight and colour vision. For colour discrimination, most mammals possess two cone populations with two visual pigments (opsins that have absorption maxima at short wavelengths (blue or ultraviolet light and long wavelengths (green or red light. Microchiropteran bats, which use echolocation to navigate and forage in complete darkness, have long been considered to have pure rod retinae. Here we use opsin immunohistochemistry to show that two phyllostomid microbats, Glossophaga soricina and Carollia perspicillata, possess a significant population of cones and express two cone opsins, a shortwave-sensitive (S opsin and a longwave-sensitive (L opsin. A substantial population of cones expresses S opsin exclusively, whereas the other cones mostly coexpress L and S opsin. S opsin gene analysis suggests ultraviolet (UV, wavelengths <400 nm sensitivity, and corneal electroretinogram recordings reveal an elevated sensitivity to UV light which is mediated by an S cone visual pigment. Therefore bats have retained the ancestral UV tuning of the S cone pigment. We conclude that bats have the prerequisite for daylight vision, dichromatic colour vision, and UV vision. For bats, the UV-sensitive cones may be advantageous for visual orientation at twilight, predator avoidance, and detection of UV-reflecting flowers for those that feed on nectar.

  20. On Dispersion in Visual Photoreceptors

    NARCIS (Netherlands)

    Stavenga, D.G.; Barneveld, H.H. van

    1975-01-01

    An idealized visual pigment absorbance spectrum is used together with a Kramers-Kronig dispersion relation to calculate the contribution of the visual pigment to the refractive index of the fly photoreceptor. It appears that an absorption coefficient of 0.010 µm-1 results in a refractive index varia

  1. Photoreceptors: unconventional ways of seeing

    OpenAIRE

    Diaz, Naryttza N.; Sprecher, Simon G.

    2011-01-01

    Animals perceive light typically by photoreceptor neurons assembled in eyes, but some also use non-eye photosensory neurons. Multidendritic neurons in the body wall of Drosophila larvae have now been shown to use an unconventional phototransduction mechanism to sense light.

  2. Photoreceptor proteins from purple bacteria

    NARCIS (Netherlands)

    Hendriks, J.; van der Horst, M.A.; Chua, T.K.; Ávila Pérez, M.; van Wilderen, L.J.; Alexandre, M.T.A.; Groot, M.-L.; Kennis, J.T.M.; Hellingwerf, K.J.; Hunter, C.N.; Daldal, F.; Thurnauer, M.C.; Beatty, J.T.

    2009-01-01

    Purple bacteria contain representatives of four of the six main families of photoreceptor proteins: phytochromes, BLUF domain containing proteins, xanthopsins (i.e., photoactive yellow proteins), and phototropins (containing one or more light, oxygen, or voltage (LOV) domains). Most of them have a

  3. Defects in RGS9 or its anchor protein R9AP in patients with slow photoreceptor deactivation

    NARCIS (Netherlands)

    Nishiguchi, KM; Sandberg, MA; Kooijman, AC; Martemyanov, KA; Pott, JWR; Hagstrom, SA; Arshavsky, VY; Berson, EL; Dryja, TP

    2004-01-01

    The RGS proteins are GTPase activating proteins that accelerate the deactivation of G proteins in a variety of signalling pathways in eukaryotes(1-6). RGS9 deactivates the G proteins (transducins) in the rod and cone phototransduction cascades(7,8). It is anchored to photoreceptor membranes by the t

  4. Photoreceptor Differentiation following Transplantation of Allogeneic Retinal Progenitor Cells to the Dystrophic Rhodopsin Pro347Leu Transgenic Pig

    DEFF Research Database (Denmark)

    Klassen, H; Kiilgaard, Jens Folke; Warfvinge, K;

    2012-01-01

    Purpose. Transplantation of stem, progenitor, or precursor cells has resulted in photoreceptor replacement and evidence of functional efficacy in rodent models of retinal degeneration. Ongoing work has been directed toward the replication of these results in a large animal model, namely, the pig....... Methods. Retinal progenitor cells were derived from the neural retina of GFP-transgenic pigs and transplanted to the subretinal space of rhodopsin Pro347Leu-transgenic allorecipients, in the early stage of the degeneration and the absence of immune suppression. Results. Results confirm the survival...... of allogeneic porcine RPCs without immune suppression in the setting of photoreceptor dystrophy. The expression of multiple photoreceptor markers by grafted cells included the rod outer segment-specific marker ROM-1. Further evidence of photoreceptor differentiation included the presence of numerous...

  5. Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.

    Science.gov (United States)

    Nikonov, Sergei S; Daniele, Lauren L; Zhu, Xuemei; Craft, Cheryl M; Swaroop, Anand; Pugh, Edward N

    2005-03-01

    The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl-/-) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl-/- mice were recorded with suction pipettes at 35-37 degrees C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette ("OS in"), or in a novel configuration with a portion of the inner segment drawn in ("OS out"). Nrl-/- photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses tpeak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, tau(D) = 110 ms vs. 240 ms, respectively. Nrl-/- photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl-/- outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl-/- cell were driven by both the short-wave (S, lambda(max) approximately 360 nm) and the mid-wave (M, lambda(max) approximately 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl-/-/Grk1-/- photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to

  6. Genetic Dissection of Dual Roles for the Transcription Factor six7 in Photoreceptor Development and Patterning in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Mailin Sotolongo-Lopez

    2016-04-01

    Full Text Available The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7 regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljrp23ahub. Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation

  7. Genetic Dissection of Dual Roles for the Transcription Factor six7 in Photoreceptor Development and Patterning in Zebrafish.

    Science.gov (United States)

    Sotolongo-Lopez, Mailin; Alvarez-Delfin, Karen; Saade, Carole J; Vera, Daniel L; Fadool, James M

    2016-04-01

    The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7) regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub) mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljrp23ahub. Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation across species.

  8. Sumoylation of bZIP transcription factor NRL modulates target gene expression during photoreceptor differentiation.

    Science.gov (United States)

    Roger, Jerome E; Nellissery, Jacob; Kim, Douglas S; Swaroop, Anand

    2010-08-13

    Development of rod photoreceptors in the mammalian retina is critically dependent on the basic motif-leucine zipper transcription factor NRL (neural retina leucine zipper). In the absence of NRL, photoreceptor precursors in mouse retina produce only cones that primarily express S-opsin. Conversely, ectopic expression of NRL in post-mitotic precursors leads to a rod-only retina. To explore the role of signaling molecules in modulating NRL function, we identified putative sites of post-translational modification in the NRL protein by in silico analysis. Here, we demonstrate the sumoylation of NRL in vivo and in vitro, with two small ubiquitin-like modifier (SUMO) molecules attached to the Lys-20 residue. NRL-K20R and NRL-K20R/K24R sumoylation mutants show reduced transcriptional activation of Nr2e3 and rhodopsin promoters (two direct targets of NRL) in reporter assays when compared with wild-type NRL. Consistent with this, in vivo electroporation of the NRL-K20R/K24R mutant into newborn Nrl(-/-) mouse retina leads to reduced Nr2e3 activation and only a partial rescue of the Nrl(-/-) phenotype in contrast to the wild-type NRL that is able to convert cones to rod photoreceptors. Although PIAS3 (protein inhibitor of activated STAT3), an E3-SUMO ligase implicated in photoreceptor differentiation, can be immunoprecipitated with NRL, there appears to be redundancy in E3 ligases, and PIAS3 does not seem to be essential for NRL sumoylation. Our studies suggest an important role of sumoylation in fine-tuning the activity of NRL and thereby incorporating yet another layer of control in gene regulatory networks involved in photoreceptor development and homeostasis.

  9. Sumoylation of bZIP Transcription Factor NRL Modulates Target Gene Expression during Photoreceptor Differentiation*

    Science.gov (United States)

    Roger, Jerome E.; Nellissery, Jacob; Kim, Douglas S.; Swaroop, Anand

    2010-01-01

    Development of rod photoreceptors in the mammalian retina is critically dependent on the basic motif-leucine zipper transcription factor NRL (neural retina leucine zipper). In the absence of NRL, photoreceptor precursors in mouse retina produce only cones that primarily express S-opsin. Conversely, ectopic expression of NRL in post-mitotic precursors leads to a rod-only retina. To explore the role of signaling molecules in modulating NRL function, we identified putative sites of post-translational modification in the NRL protein by in silico analysis. Here, we demonstrate the sumoylation of NRL in vivo and in vitro, with two small ubiquitin-like modifier (SUMO) molecules attached to the Lys-20 residue. NRL-K20R and NRL-K20R/K24R sumoylation mutants show reduced transcriptional activation of Nr2e3 and rhodopsin promoters (two direct targets of NRL) in reporter assays when compared with wild-type NRL. Consistent with this, in vivo electroporation of the NRL-K20R/K24R mutant into newborn Nrl−/− mouse retina leads to reduced Nr2e3 activation and only a partial rescue of the Nrl−/− phenotype in contrast to the wild-type NRL that is able to convert cones to rod photoreceptors. Although PIAS3 (protein inhibitor of activated STAT3), an E3-SUMO ligase implicated in photoreceptor differentiation, can be immunoprecipitated with NRL, there appears to be redundancy in E3 ligases, and PIAS3 does not seem to be essential for NRL sumoylation. Our studies suggest an important role of sumoylation in fine-tuning the activity of NRL and thereby incorporating yet another layer of control in gene regulatory networks involved in photoreceptor development and homeostasis. PMID:20551322

  10. Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina.

    Science.gov (United States)

    Gonzalez-Cordero, Anai; West, Emma L; Pearson, Rachael A; Duran, Yanai; Carvalho, Livia S; Chu, Colin J; Naeem, Arifa; Blackford, Samuel J I; Georgiadis, Anastasios; Lakowski, Jorn; Hubank, Mike; Smith, Alexander J; Bainbridge, James W B; Sowden, Jane C; Ali, Robin R

    2013-08-01

    Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.

  11. Retinoic Acid Protects and Rescues the Development of Zebrafish Embryonic Retinal Photoreceptor Cells from Exposure to Paclobutrazol

    Directory of Open Access Journals (Sweden)

    Wen-Der Wang

    2017-01-01

    Full Text Available Paclobutrazol (PBZ is a widely used fungicide that shows toxicity to aquatic embryos, probably through rain-wash. Here, we specifically focus on its toxic effect on eye development in zebrafish, as well as the role of retinoic acid (RA, a metabolite of vitamin A that controls proliferation and differentiation of retinal photoreceptor cells, in this toxicity. Embryos were exposed to PBZ with or without RA from 2 to 72 h post-fertilization (hpf, and PBZ-treated embryos (2–72 hpf were exposed to RA for additional hours until 120 hpf. Eye size and histology were examined. Expression levels of gnat1 (rod photoreceptor marker, gnat2 (cone photoreceptor marker, aldehyde dehydrogenases (encoding key enzymes for RA synthesis, and phospho-histone H3 (an M-phase marker in the eyes of control and treated embryos were examined. PBZ exposure dramatically reduces photoreceptor proliferation, thus resulting in a thinning of the photoreceptor cell layer and leading to a small eye. Co-treatment of PBZ with RA, or post-treatment of PBZ-treated embryos with RA, partially rescues photoreceptor cells, revealed by expression levels of marker proteins and by retinal cell proliferation. PBZ has strong embryonic toxicity to retinal photoreceptors, probably via suppressing the production of RA, with effects including impaired retinal cell division.

  12. Estimating photoreceptor excitations from spectral outputs of a personal light exposure measurement device.

    Science.gov (United States)

    Cao, Dingcai; Barrionuevo, Pablo A

    2015-03-01

    The intrinsic circadian clock requires photoentrainment to synchronize the 24-hour solar day. Therefore, light stimulation is an important component of chronobiological research. Currently, the chronobiological research field overwhelmingly uses photopic illuminance that is based on the luminous efficiency function, V(λ), to quantify light levels. However, recent discovery of intrinsically photosensitive retinal ganglion cells (ipRGCs), which are activated by self-contained melanopsin photopigment and also by inputs from rods and cones, makes light specification using a one-dimensional unit inadequate. Since the current understanding of how different photoreceptor inputs contribute to the circadian system through ipRGCs is limited, it is recommended to specify light in terms of the excitations of five photoreceptors (S-, M-, L-cones, rods and ipRGCs; Lucas et al., 2014). In the current study, we assessed whether the spectral outputs from a commercially available spectral watch (i.e. Actiwatch Spectrum) could be used to estimate photoreceptor excitations. Based on the color sensor spectral sensitivity functions from a previously published work, as well as from our measurements, we computed spectral outputs in the long-wavelength range (R), middle-wavelength range (G), short-wavelength range (B) and broadband range (W) under 52 CIE illuminants (25 daylight illuminants, 27 fluorescent lights). We also computed the photoreceptor excitations for each illuminant using human photoreceptor spectral sensitivity functions. Linear regression analyses indicated that the Actiwatch spectral outputs could predict photoreceptor excitations reliably, under the assumption of linear responses of the Actiwatch color sensors. In addition, R, G, B outputs could classify illuminant types (fluorescent versus daylight illuminants) satisfactorily. However, the assessment of actual Actiwatch recording under several testing light sources showed that the spectral outputs were subject to

  13. Design of CGMP production of 18F- and 68Ga-radiopharmaceuticals.

    Science.gov (United States)

    Chi, Yen-Ting; Chu, Pei-Chun; Chao, Hao-Yu; Shieh, Wei-Chen; Chen, Chuck C

    2014-01-01

    Radiopharmaceutical production process must adhere to current good manufacturing process (CGMP) compliance to ensure the quality of precursor, prodrug (active pharmaceutical ingredient, API), and the final drug product that meet acceptance criteria. We aimed to develop an automated system for production of CGMP grade of PET radiopharmaceuticals. The hardware and software of the automated synthesizer that fit in the hot cell under cGMP requirement were developed. Examples of production yield and purity for (68)Ga-DOTATATE and (18)F-FDG at CGMP facility were optimized. Analytical assays and acceptance criteria for cGMP grade of (68)Ga-DOTATATE and (18)F-FDG were established. CGMP facility for the production of PET radiopharmaceuticals has been established. Radio-TLC and HPLC analyses of (68)Ga-DOTATATE and (18)F-FDG showed that the radiochemical purity was 92% and 96%, respectively. The products were sterile and pyrogenic-free. CGMP compliance of radiopharmaceuticals has been reviewed. (68)Ga-DOTATATE and (18)F-FDG were synthesized with high radiochemical yield under CGMP process.

  14. Programming Retinal Stem Cells into Cone Photoreceptors

    Science.gov (United States)

    2015-12-01

    this grant, we sought to investigate the mechanisms that regulate the earliest events in cone photoreceptor development and to exploit this knowledge...the mRNA for three transcription factors promoted cone photoreceptor formation in retinal stem cells derived from human embryonic stem cells. These...reverse vision loss. 15. SUBJECT TERMS Cone photoreceptor, retina, retinal stem cell, Otx2, Onecut1, Blimp1, RNA-seq., transcription factors, and

  15. Photoreceptor damage following exposure to excess riboflavin.

    Science.gov (United States)

    Eckhert, C D; Hsu, M H; Pang, N

    1993-12-15

    Flavins generate oxidants during metabolism and when exposed to light. Here we report that the photoreceptor layer of retinas from black-eyed rats is reduced in size by a dietary regime containing excess riboflavin. The effect of excess riboflavin was dose-dependent and was manifested by a decrease in photoreceptor length. This decrease was due in part to a reduction in the thickness of the outer nuclear layer, a structure formed from stacked photoreceptor nuclei. These changes were accompanied by an increase in photoreceptor outer segment autofluorescence following illumination at 328 nm, a wavelength that corresponds to the excitation maxima of oxidized lipopigments of the retinal pigment epithelium.

  16. Müller glial cells induce stem cell properties in retinal progenitors in vitro and promote their further differentiation into photoreceptors.

    Science.gov (United States)

    Simón, María V; De Genaro, Pablo; Abrahan, Carolina E; de los Santos, Beatriz; Rotstein, Nora P; Politi, Luis E

    2012-02-01

    Using stem cells to replace lost neurons is a promising strategy for treating retinal neurodegenerative diseases. Among their multiple functions, Müller glial cells are retina stem cells, with a robust regenerative potential in lower vertebrates, which is much more restricted in mammals. In rodents, most retina progenitors exit the cell cycle immediately after birth, differentiate as neurons, and then cannot reenter the cell cycle. Here we demonstrate that, in mixed cultures with Müller glial cells, rat retina progenitor cells expressed stem cell properties, maintained their proliferative potential, and were able to preserve these properties and remain mitotically active after several consecutive passages. Notably, these progenitors retained the capacity to differentiate as photoreceptors, even after successive reseedings. Müller glial cells markedly stimulated differentiation of retina progenitors; these cells initially expressed Crx and then developed as mature photoreceptors that expressed characteristic markers, such as opsin and peripherin. Moreover, they were light responsive, insofar as they decreased their cGMP levels when exposed to light, and they also showed high-affinity glutamate uptake, a characteristic of mature photoreceptors. Our present findings indicate that, in addition to giving rise to new photoreceptors, Müller glial cells might instruct a pool of undifferentiated cells to develop and preserve stem cell characteristics, even after successive reseedings, and then stimulate their differentiation as functional photoreceptors. This complementary mechanism might contribute to enlarge the limited regenerative capacity of mammalian Müller cells.

  17. Tie rod insertion test

    CERN Multimedia

    B. LEVESY

    2002-01-01

    The superconducting coil is inserted in the outer vaccum tank and supported by a set of tie rods. These tie rods are made of titanium alloy. This test reproduce the final insertion of the tie rods inside the outer vacuum tank.

  18. Derivation of neurons with functional properties from adult limbal epithelium: implications in autologous cell therapy for photoreceptor degeneration.

    Science.gov (United States)

    Zhao, Xing; Das, Ani V; Bhattacharya, Sumitra; Thoreson, Wallace B; Sierra, Jorge Rodriguez; Mallya, Kavita B; Ahmad, Iqbal

    2008-04-01

    The limbal epithelium (LE), a circular and narrow epithelium that separates cornea from conjunctiva, harbors stem cells/progenitors in its basal layer that regenerate cornea. We have previously demonstrated that cells in the basal LE, when removed from their niche and cultured in reduced bond morphogenetic protein signaling, acquire properties of neural progenitors. Here, we demonstrate that LE-derived neural progenitors generate neurons with functional properties and can be directly differentiated along rod photoreceptor lineage in vitro and in vivo. These observations posit the LE as a potential source of neural progenitors for autologous cell therapy to treat photoreceptor degeneration in age-related macular degeneration and retinitis pigmentosa.

  19. The Transcription Factor Neural Retina Leucine Zipper (NRL) Controls Photoreceptor-specific Expression of Myocyte Enhancer Factor Mef2c from an Alternative Promoter*

    Science.gov (United States)

    Hao, Hong; Tummala, Padmaja; Guzman, Eduardo; Mali, Raghuveer S.; Gregorski, Janina; Swaroop, Anand; Mitton, Kenneth P.

    2011-01-01

    Neural retina leucine zipper (NRL) is an essential transcription factor for cell fate specification and functional maintenance of rod photoreceptors in the mammalian retina. In the Nrl−/− mouse retina, photoreceptor precursors fail to produce rods and generate functional cone photoreceptors that predominantly express S-opsin. Previous global expression analysis using microarrays revealed dramatically reduced expression of myocyte enhancer factor Mef2c in the adult Nrl−/− retina. We undertook this study to examine the biological relevance of Mef2c expression in retinal rod photoreceptors. Bioinformatics analysis, rapid analysis of cDNA ends (5′-RACE), and reverse transcription coupled with qPCR using splice site-specific oligonucleotides suggested that Mef2c is expressed in the mature retina from an alternative promoter. Chromatin immunoprecipitation (ChIP) studies showed the association of active RNA polymerase II and acetylated histone H3 just upstream of Mef2c exon 4, providing additional evidence for the utilization of an alternative promoter in the retina. In concordance, we observed the binding of NRL to a putative NRL-response element (NRE) at this location by ChIP-seq and electrophoretic mobility shift assays. NRL also activated the Mef2c alternative promoter in vitro and in vivo. Notably, MEF2C could support Rhodopsin promoter activity in rod photoreceptors. We conclude that Mef2c expression from an alternative promoter in the retina is regulated by NRL. Our studies also implicate MEF2C as a transcriptional regulator of homeostasis in rod photoreceptor cells. PMID:21849497

  20. Insights into the role of RD3 in guanylate cyclase trafficking, photoreceptor degeneration and Leber Congenital Amaurosis

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    Robert S. Molday

    2014-05-01

    Full Text Available RD3 is an evolutionarily conserved 23 kDa protein expressed in rod and cone photoreceptor cells. Mutations in the gene encoding RD3 resulting in unstable non-functional C-terminal truncated proteins are responsible for early onset photoreceptor degeneration in Leber Congenital Amaurosis 12 (LCA12 patients, the rd3 mice, and the rcd2 collies. Recent studies have shown that RD3 interacts with guanylate cyclases GC1 and GC2 in retinal cell extracts and HEK293 cells co-expressing GC and RD3. This interaction inhibits GC catalytic activity and promotes the exit of GC1 and GC2 from the endoplasmic reticulum and their trafficking to photoreceptor outer segments. Adeno-associated viral vector delivery of the normal RD3 gene to photoreceptors of the Rd3 mouse restores GC1 and GC2 expression and outer segment localization and leads to the long-term recovery of visual function and photoreceptor cell survival. This review focuses on the genetic and biochemical studies that have provided insight into the role of RD3 in photoreceptor function and survival.

  1. Retbindin is an extracellular riboflavin-binding protein found at the photoreceptor/retinal pigment epithelium interface.

    Science.gov (United States)

    Kelley, Ryan A; Al-Ubaidi, Muayyad R; Naash, Muna I

    2015-02-20

    Retbindin is a novel retina-specific protein of unknown function. In this study, we have used various approaches to evaluate protein expression, localization, biochemical properties, and function. We find that retbindin is secreted by the rod photoreceptors into the inter-photoreceptor matrix where it is maintained via electrostatic forces. Retbindin is predominantly localized at the interface between photoreceptors and retinal pigment epithelium microvilli, a region critical for retinal function and homeostasis. Interestingly, although it is associated with photoreceptor outer segments, retbindin's expression is not dependent on their presence. In vitro, retbindin is capable of binding riboflavin, thus implicating the protein as a metabolite carrier between the retina and the retinal pigment epithelium. Altogether, our data show that retbindin is a novel photoreceptor-specific protein with a unique localization and function. We hypothesize that retbindin is an excellent candidate for binding retinal flavins and possibly participating in their transport from the extracellular space to the photoreceptors. Further investigations are warranted to determine the exact function of retbindin in retinal homeostasis and disease.

  2. Transcription coactivators p300 and CBP are necessary for photoreceptor-specific chromatin organization and gene expression.

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    Anne K Hennig

    Full Text Available Rod and cone photoreceptor neurons in the mammalian retina possess specialized cellular architecture and functional features for converting light to a neuronal signal. Establishing and maintaining these characteristics requires appropriate expression of a specific set of genes, which is tightly regulated by a network of photoreceptor transcription factors centered on the cone-rod homeobox protein CRX. CRX recruits transcription coactivators p300 and CBP to acetylate promoter-bound histones and activate transcription of target genes. To further elucidate the role of these two coactivators, we conditionally knocked out Ep300 and/or CrebBP in differentiating rods or cones, using opsin-driven Cre recombinase. Knockout of either factor alone exerted minimal effects, but loss of both factors severely disrupted target cell morphology and function: the unique nuclear chromatin organization seen in mouse rods was reversed, accompanied by redistribution of nuclear territories associated with repressive and active histone marks. Transcription of many genes including CRX targets was severely impaired, correlating with reduced histone H3/H4 acetylation (the products of p300/CBP on target gene promoters. Interestingly, the presence of a single wild-type allele of either coactivator prevented many of these defects, with Ep300 more effective than Cbp. These results suggest that p300 and CBP play essential roles in maintaining photoreceptor-specific structure, function and gene expression.

  3. Cyclic nucleotide specificity of the activator and catalytic sites of a cGMP-stimulated cGMP phosphodiesterase from Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Baraniak, Janina; Bulgakov, Roman; Jastorff, Bernd; Morr, Michael; Petridis, Georg; Stec, Wojciech J.; Seela, Frank; Haastert, Peter J.M. van

    1985-01-01

    The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant-mediated elevations of cGMP levels. The interaction of 20 cGMP derivativ

  4. Correlative intravital imaging of cGMP signals and vasodilation in mice

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    Martin eThunemann

    2014-10-01

    Full Text Available Cyclic guanosine monophosphate (cGMP is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1 epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2 ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to ‘watch’ biochemistry, (patho physiology, and pharmacotherapy in the context of a living mammalian organism.

  5. Correlative intravital imaging of cGMP signals and vasodilation in mice

    Science.gov (United States)

    Thunemann, Martin; Schmidt, Kjestine; de Wit, Cor; Han, Xiaoxing; Jain, Rakesh K.; Fukumura, Dai; Feil, Robert

    2014-01-01

    Cyclic guanosine monophosphate (cGMP) is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO)-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET)-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1) epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2) ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to “watch” biochemistry, (patho-)physiology, and pharmacotherapy in the context of a living mammalian organism. PMID:25352809

  6. Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions.

    Science.gov (United States)

    Koch, K W; Stryer, L

    1988-07-07

    Visual excitation in retinal rod cells is mediated by a cascade that leads to the amplified hydrolysis of cyclic GMP (cGMP) and the consequent closure of cGMP-activated cation-specific channels in the plasma membrane. Recovery of the dark state requires the resynthesis of cGMP, which is catalysed by guanylate cyclase, an axoneme-associated enzyme. The lowering of the cytosolic calcium concentration (Cai) following illumination is thought to be important in stimulating cyclase activity. This hypothesis is supported by the finding that the cGMP content of rod outer segments increases several-fold when Cai is lowered to less than 10 nM. It is evident that cGMP and Cai levels are reciprocally controlled by negative feedback. Guanylate cyclase from toad ROS is strongly stimulated when the calcium level is lowered from 10 microM to 10 nM, but only if they are excited by light. We show here that the guanylate cyclase activity of unilluminated bovine rod outer segments increases markedly (5 to 20-fold) when the calcium level is lowered from 200 nM to 50 nM. This steep dependence of guanylate cyclase activity on the calcium level in the physiological range has a Hill coefficient of 3.9. Stimulation at low calcium levels is mediated by a protein that can be released from the outer segment membranes by washing with a low salt buffer. Calcium sensitivity is partially restored by adding the soluble extract back to the washed membranes. The highly cooperative activation of guanylate cyclase by the light-induced lowering of Cai is likely to be a key event in restoring the dark current after excitation.

  7. Genomic evidence for rod monochromacy in sloths and armadillos suggests early subterranean history for Xenarthra.

    Science.gov (United States)

    Emerling, Christopher A; Springer, Mark S

    2015-02-07

    Rod monochromacy is a rare condition in vertebrates characterized by the absence of cone photoreceptor cells. The resulting phenotype is colourblindness and low acuity vision in dim-light and blindness in bright-light conditions. Early reports of xenarthrans (armadillos, sloths and anteaters) suggest that they are rod monochromats, but this has not been tested with genomic data. We searched the genomes of Dasypus novemcinctus (nine-banded armadillo), Choloepus hoffmanni (Hoffmann's two-toed sloth) and Mylodon darwinii (extinct ground sloth) for retinal photoreceptor genes and examined them for inactivating mutations. We performed PCR and Sanger sequencing on cone phototransduction genes of 10 additional xenarthrans to test for shared inactivating mutations and estimated the timing of inactivation for photoreceptor pseudogenes. We concluded that a stem xenarthran became an long-wavelength sensitive-cone monochromat following a missense mutation at a critical residue in SWS1, and a stem cingulate (armadillos, glyptodonts and pampatheres) and stem pilosan (sloths and anteaters) independently acquired rod monochromacy early in their evolutionary history following the inactivation of LWS and PDE6C, respectively. We hypothesize that rod monochromacy in armadillos and pilosans evolved as an adaptation to a subterranean habitat in the early history of Xenarthra. The presence of rod monochromacy has major implications for understanding xenarthran behavioural ecology and evolution.

  8. The Ciliopathy Gene ahi1 Is Required for Zebrafish Cone Photoreceptor Outer Segment Morphogenesis and Survival

    Science.gov (United States)

    Lessieur, Emma M.; Fogerty, Joseph; Gaivin, Robert J.; Song, Ping; Perkins, Brian D.

    2017-01-01

    Purpose Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of JBTS patients exhibit retinal dystrophy and/or kidney disease. Mutations in the AHI1 gene are causative for approximately 10% of all JBTS cases. The purpose of this study was to generate ahi1 mutant alleles in zebrafish and to characterize the retinal phenotypes. Methods Zebrafish ahi1 mutants were generated using transcription activator-like effector nucleases (TALENs). Expression analysis was performed by whole-mount in situ hybridization. Anatomic and molecular characterization of photoreceptors was investigated by histology, electron microscopy, and immunohistochemistry. The optokinetic response (OKR) behavior assay was used to assess visual function. Kidney cilia were evaluated by whole-mount immunostaining. Results The ahi1lri46 mutation in zebrafish resulted in shorter cone outer segments but did not affect visual behavior at 5 days after fertilization (dpf). No defects in rod morphology or rhodopsin localization were observed at 5 dpf. By 5 months of age, cone degeneration and rhodopsin mislocalization in rod photoreceptors was observed. The connecting cilium formed normally and Cc2d2a and Cep290 localized properly. Distal pronephric duct cilia were absent in mutant fish; however, only 9% of ahi1 mutants had kidney cysts by 5 dpf, suggesting that the pronephros remained largely functional. Conclusions The results indicate that Ahi1 is required for photoreceptor disc morphogenesis and outer segment maintenance in zebrafish. PMID:28118669

  9. Förster resonance energy transfer as a tool to study photoreceptor biology

    Science.gov (United States)

    Hovan, Stephanie C.; Howell, Scott; Park, Paul S.-H.

    2010-11-01

    Vision is initiated in photoreceptor cells of the retina by a set of biochemical events called phototransduction. These events occur via coordinated dynamic processes that include changes in secondary messenger concentrations, conformational changes and post-translational modifications of signaling proteins, and protein-protein interactions between signaling partners. A complete description of the orchestration of these dynamic processes is still unavailable. Described in this work is the first step in the development of tools combining fluorescent protein technology, Förster resonance energy transfer (FRET), and transgenic animals that have the potential to reveal important molecular insights about the dynamic processes occurring in photoreceptor cells. We characterize the fluorescent proteins SCFP3A and SYFP2 for use as a donor-acceptor pair in FRET assays, which will facilitate the visualization of dynamic processes in living cells. We also demonstrate the targeted expression of these fluorescent proteins to the rod photoreceptor cells of Xenopus laevis, and describe a general method for detecting FRET in these cells. The general approaches described here can address numerous types of questions related to phototransduction and photoreceptor biology by providing a platform to visualize dynamic processes in molecular detail within a native context.

  10. Förster resonance energy transfer as a tool to study photoreceptor biology.

    Science.gov (United States)

    Hovan, Stephanie C; Howell, Scott; Park, Paul S-H

    2010-01-01

    Vision is initiated in photoreceptor cells of the retina by a set of biochemical events called phototransduction. These events occur via coordinated dynamic processes that include changes in secondary messenger concentrations, conformational changes and post-translational modifications of signaling proteins, and protein-protein interactions between signaling partners. A complete description of the orchestration of these dynamic processes is still unavailable. Described in this work is the first step in the development of tools combining fluorescent protein technology, Förster resonance energy transfer (FRET), and transgenic animals that have the potential to reveal important molecular insights about the dynamic processes occurring in photoreceptor cells. We characterize the fluorescent proteins SCFP3A and SYFP2 for use as a donor-acceptor pair in FRET assays, which will facilitate the visualization of dynamic processes in living cells. We also demonstrate the targeted expression of these fluorescent proteins to the rod photoreceptor cells of Xenopus laevis, and describe a general method for detecting FRET in these cells. The general approaches described here can address numerous types of questions related to phototransduction and photoreceptor biology by providing a platform to visualize dynamic processes in molecular detail within a native context.

  11. Abnormal photoreceptor outer segment development and early retinal degeneration in kif3a mutant zebrafish.

    Science.gov (United States)

    Raghupathy, Rakesh K; Zhang, Xun; Alhasani, Reem H; Zhou, Xinzhi; Mullin, Margaret; Reilly, James; Li, Wenchang; Liu, Mugen; Shu, Xinhua

    2016-08-01

    Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A (kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport.

  12. Role of spectraplakin in Drosophila photoreceptor morphogenesis.

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    Uyen Ngoc Mui

    Full Text Available BACKGROUND: Crumbs (Crb, a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors. METHODOLOGY/PRINCIPAL FINDINGS: The localization pattern of Spectraplakin in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin's gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test. CONCLUSIONS/SIGNIFICANCE: In summary, we found a unique localization of Spectraplakin in photoreceptors, and identified the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions through genetic mutational analysis. Our data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis.

  13. FIZ1 is part of the regulatory protein complex on active photoreceptor-specific gene promoters in vivo

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    Chen Shiming

    2008-10-01

    Full Text Available Abstract Background FIZ1 (Flt-3 Interacting Zinc-finger is a broadly expressed protein of unknown function. We reported previously that in the mammalian retina, FIZ1 interacts with NRL (Neural-Retina Leucine-zipper, an essential transcriptional activator of rod photoreceptor-specific genes. The concentration of FIZ1 in the retina increases during photoreceptor terminal maturation, when two key transcription factors NRL and CRX (Cone-Rod Homeobox become detectable on the promoters of photoreceptor-specific genes (i.e. Rhodopsin, Pde6b. To determine if FIZ1 is involved in regulating CRX-mediated transcriptional activation, we examined FIZ1 subcellular location in mouse neural retina, its ability to interact with CRX, and its association with CRX/NRL target genes. Results FIZ1 is present in the nucleus of adult photoreceptors as well as other retinal neurons as shown by transmission electron microscopy with nano-gold labeling. FIZ1 and CRX were co-precipitated from retinal nuclear extracts with antibodies to either protein. Chromatin immunoprecipitation (ChIP assays revealed that FIZ1 is part of the protein complex on several rod and cone gene promoters, within photoreceptor cells of the mouse retina. FIZ1 complexes with CRX or NRL on known NRL- and CRX-responsive elements, as shown by electrophoretic mobility shift assays with FIZ1 antibody. FIZ1 can directly bind to CRX, as demonstrated using yeast two-hybrid and GST pull-down assays. Co-transfection assays demonstrated that FIZ1 increases CRX-mediated activation of Opsin test promoters. Quantitative ChIP analysis revealed an increased association of FIZ1 with the Rhodopsin promoter in adult (P-25 neural retina versus immature (P-3 neural retina. The quantity of transcriptionally active RNA Polymerase-II within the Rhodopsin gene (Rho was significantly increased in the adult neural retina, compared to the immature retina. Conclusion FIZ1 directly interacts with CRX to enhance CRX

  14. Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods.

    Science.gov (United States)

    Frederiksen, Rikard; Boyer, Nicholas P; Nickle, Benjamin; Chakrabarti, Kalyan S; Koutalos, Yiannis; Crouch, Rosalie K; Oprian, Daniel; Cornwall, M Carter

    2012-06-01

    We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.

  15. Telescopic drilling rod

    Energy Technology Data Exchange (ETDEWEB)

    Kagan, I.L.; Berezov, S.I.; Gavrilov, G.A.; Goykhman, Ya.A.; Makushkin, D.O.; Rachev, M.P.; Voynich, L.K.

    1981-09-07

    The telescopic drilling rod includes an inner section of the rod, in whose center cable has been passed and is attached a bearing assembly connecting it to the winch, outer section of rod along which there is pipeline connecting the working cavity formed by the inner section of rod and the housing, installed on the lower end of the outer section of rod, with cavity formed by framework of the guide swivel and end piece and connected to the hydraulic system of the machine by pipeline, as well as clamping elements. In order to drill wells to a depth greater than the length of the outer sectrion of the rod, the latter jointly with the inner section of rod is lowered into the extreme lower position until swivel rests on the feed mechanism. With further slipping of cable and the absence of pressure in the hydraulic system, clamping elements do not have an effect on the inner section of rod. It has the opportunity to freely move along the outer section of rod downwards to the face. When pressure is supplied on pipeline into cavity and further through pipeline into working cavity, the inner section of rod is clamped with feed of the outer section in the process of drilling, both sections move jointly. Because of the link between working cavity of sleeve installed on the lower end of the outer section of rod, and the hydraulic system of the machine through the swivel cavity, it is possible to fix the drilling rod in any mutual axial position of the section.

  16. Absorption spectra and linear dichroism of some amphibian photoreceptors.

    Science.gov (United States)

    Hárosi, F I

    1975-09-01

    Absorption spectra and linear dichroism of dark-adapted, isolated photoreceptors of mudpuppies, larval and adult tiger salamanders, and tropical toads were measured microspectrophotometrically. Spectral half-band width, dichroic ratio, and transverse specific density were determined using averaged polarized absorptance spectra and photomicrographs of seven types of rod outer segments. Two classes of cells were found, one with higher specific density and dichroic ratio, associable with the presence of rhodopsins, the other, lower in both quantities, associable with porphyropsins. Relationships were derived to calculate the product of molar concentration and extinction coefficient (CEmax) from specific density and dichroic ratio. By utilizing the hypothesis of invariance of oscillator strengths and measured half-band widths, Emax values were independently determined, permitting the calculation of C. The pigment concentration for all cells tested was about 3.5 mM. The broadness of green rod pigment spectra is correlated with reduced molar absorptivity and reduced cellular specific density. Estimation of physiological spectral sensitivities is discussed. Based on dichroic ratio considerations, a model is proposed for the orientation of retinals in situ which could account for the apparent degree of alignment of transition moments. In the chosen orientation, the ring portion of conjugation becomes primarily responsible for axial extinction. Reduced dichroism of dehydroretinal-bearing cells can thus result from the extended ring conjugation of chromophores. Some inferences derivable from the model are discussed.

  17. Development of a whole cell pneumococcal vaccine: BPL inactivation, cGMP production, and stability.

    Science.gov (United States)

    Gonçalves, Viviane M; Dias, Waldely O; Campos, Ivana B; Liberman, Celia; Sbrogio-Almeida, Maria E; Silva, Eliane P; Cardoso, Celso P; Alderson, Mark; Robertson, George; Maisonneuve, Jean-François; Tate, Andrea; Anderson, Porter; Malley, Richard; Fratelli, Fernando; Leite, Luciana C C

    2014-02-19

    Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine. Initially, three engineering runs and two cGMP runs were performed in 60-L bioreactors, demonstrating the consistency of the production process, as evaluated by the growth curves, glucose consumption and metabolite formation (lactate and acetate). Cell recovery by tangential filtration was 92 ± 13 %. We optimized the conditions for beta-propiolactone (BPL) inactivation of the bacterial suspensions, establishing a maximum cell density of OD600 between 27 and 30, with a BPL concentration of 1:4000 (v/v) at 150 rpm and 4 °C for 30 h. BPL was hydrolyzed by heating for 2h at 37 °C. The criteria and methods for quality control were defined using the engineering runs and the cGMP Lots passed all specifications. cGMP vaccine Lots displayed high potency, inducing between 80 and 90% survival in immunized mice when challenged with virulent pneumococci. Sera from mice immunized with the cGMP Lots recognized several pneumococcal proteins in the extract of encapsulated strains by Western blot. The cGMP whole cell antigen bulk and whole cell vaccine product lots were shown to be stable for up to 12 and 18 months, respectively, based upon survival assays following i.p. challenge. Our results show the consistency and stability of the cGMP whole cell pneumococcal vaccine lots and demonstrate the feasibility of production in a developing country setting.

  18. Machine learning approaches to supporting the identification of photoreceptor-enriched genes based on expression data

    Directory of Open Access Journals (Sweden)

    Simpson David

    2006-03-01

    Full Text Available Abstract Background Retinal photoreceptors are highly specialised cells, which detect light and are central to mammalian vision. Many retinal diseases occur as a result of inherited dysfunction of the rod and cone photoreceptor cells. Development and maintenance of photoreceptors requires appropriate regulation of the many genes specifically or highly expressed in these cells. Over the last decades, different experimental approaches have been developed to identify photoreceptor enriched genes. Recent progress in RNA analysis technology has generated large amounts of gene expression data relevant to retinal development. This paper assesses a machine learning methodology for supporting the identification of photoreceptor enriched genes based on expression data. Results Based on the analysis of publicly-available gene expression data from the developing mouse retina generated by serial analysis of gene expression (SAGE, this paper presents a predictive methodology comprising several in silico models for detecting key complex features and relationships encoded in the data, which may be useful to distinguish genes in terms of their functional roles. In order to understand temporal patterns of photoreceptor gene expression during retinal development, a two-way cluster analysis was firstly performed. By clustering SAGE libraries, a hierarchical tree reflecting relationships between developmental stages was obtained. By clustering SAGE tags, a more comprehensive expression profile for photoreceptor cells was revealed. To demonstrate the usefulness of machine learning-based models in predicting functional associations from the SAGE data, three supervised classification models were compared. The results indicated that a relatively simple instance-based model (KStar model performed significantly better than relatively more complex algorithms, e.g. neural networks. To deal with the problem of functional class imbalance occurring in the dataset, two data re

  19. Optical imaging of human cone photoreceptors directly following the capture of light.

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    Phillip Bedggood

    Full Text Available Capture of light in the photoreceptor outer segment initiates a cascade of chemical events that inhibit neurotransmitter release, ultimately resulting in vision. The massed response of the photoreceptor population can be measured non-invasively by electrical recordings, but responses from individual cells cannot be measured without dissecting the retina. Here we used optical imaging to observe individual human cones in the living eye as they underwent bleaching of photopigment and associated phototransduction. The retina was simultaneously stimulated and observed with high intensity visible light at 1 kHz, using adaptive optics. There was marked variability between individual cones in both photosensitivity and pigment optical density, challenging the conventional assumption that photoreceptors act as identical subunits (coefficient of variation in rate of photoisomerization = 23%. There was also a pronounced inverse correlation between these two parameters (p<10(-7; the temporal evolution of image statistics revealed this to be a dynamic relationship, with cone waveguiding efficiency beginning a dramatic increase within 3 ms of light onset. Beginning as early as 2 ms after light onset and including half of cells by ∼7 ms, cone intensity showed reversals characteristic of interference phenomena, with greater delays in reversal corresponding to cones with more photopigment (p<10(-3. The timing of these changes is argued to best correspond with either the cessation of dark current, or to related events such as changes in intracellular cGMP. Cone intensity also showed fluctuations of high frequency (332±25 Hz and low amplitude (3.0±0.85%. Other groups have shown similar fluctuations that were directly evoked by light; if this corresponds to the same phenomenon, we propose that the amplitude of fluctuation may be increased by the use of a bright flash followed by a brief pause, to allow recovery of cone circulating current.

  20. Structure and function of the retinal pigment epithelium, photoreceptors and cornea in the eye of Sardinella aurita (Clupeidae, Teleostei

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    Mostafa Ali Salem

    2016-05-01

    Full Text Available The structure of the pigment epithelium, photoreceptors and the cornea in the eye of a teleost, Sardinella aurita was examined by light and electron microscopy. The retinal pigment epithelium forms a single layer of cells joined laterally by cell junctions. Centrally in the retina these cells are columnar, while more peripherally they become cuboidal in shape. The basal (scleral border of the pigment epithelial cells is not infolded but is relatively smooth. Phagosomes containing lysosome-like bodies are also common features of the retinal pigment epithelium. Numerous melanosomes (pigment granules are abundant throughout the epithelial cells. These melanosomes probably absorb light which has passed through the photoreceptor layer. Four photoreceptor cells were identified; rods, long single cones, short single cones and double cones. The presence of these types suggests a diversity of photoreceptor function. Square mosaic pattern of cones and well-developed choroid gland are also main features of the eye. The inner segment of rods and cones were rich in organelles indicating much synthetic activity. Calycal processes projecting from cone outer segments are also observed. The cornea includes an epithelium with a complex pattern of surface microplicae, a basement membrane, dermal stroma, an iridescent layer, scleral stroma, Descemet’s membrane and endothelium. The autochthonous layer which is seen in some teleosts has not been observed in the cornea of this species. These and other observations were discussed in relation to the photic environment and habits of this fish.

  1. Retinal photoreceptor fine structure in the red-tailed hawk (Buteo jamaicensis).

    Science.gov (United States)

    Braekevelt, C R

    1993-09-01

    The retinal photoreceptors of the red-tailed hawk (Buteo jamaicensis) consist of rods, single cones and double (unequal) cones present in a ratio of about 2:1:5. In the light-adapted state, the rods are slender elongated cells with outer segments that reach to the retinal epithelial (RPE) cells. The inner segment displays an ellipsoid of mitochondria, plentiful polysomes, some rough ER and Golgi zones. The rod nucleus is located deep within the outer nuclear layer and the synaptic spherule displays both invaginated (ribbon) and superficial (conventional) synaptic sites. Single cones show a thin tapering outer segment, a large electron lucent oil droplet at the apex of the inner segment and an ellipsoid of mitochondria. Double cones consist of a larger chief member which displays a thin tapering outer segment and an electron dense oil droplet as well as a smaller accessory cone which shows no oil droplet, an ellipsoid and a paraboloid of glycogen. As in the single cone, polysomes, RER and Golgi zones are also noted in the inner segments of both members of the double cone. Near the external limiting membrane the chief and accessory cones show membrane specializations indicative of junctions on their contiguous surfaces. All cone photoreceptors are of a smaller diameter than is normally reported for avian species. Both single and double cones display several invaginated synapses as well as numerous superficial synaptic sites.

  2. Photoreceptor types, visual pigments, and topographic specializations in the retinas of hydrophiid sea snakes.

    Science.gov (United States)

    Hart, Nathan S; Coimbra, João Paulo; Collin, Shaun P; Westhoff, Guido

    2012-04-15

    Sea snakes have evolved numerous anatomical, physiological, and behavioral adaptations to suit their wholly aquatic lifestyle. However, although sea snakes use vision for foraging and mate selection, little is known about their visual abilities. We used microspectrophotometry, light microscopy, and scanning electron microscopy to characterize the retinal photoreceptors of spine-bellied (Lapemis curtus) and horned (Acalyptophis peronii) sea snakes. Both species have three types of visual pigment sensitive to short (SWS; wavelength of maximum absorbance, λmax 428-430 nm), medium (MWS; λmax 496 nm), and long wavelengths of light (LWS; λmax 555-559 nm) in each of three different subtypes of cone-like single photoreceptor. They also possess a cone-like double photoreceptor subtype, both the principal and accessory member of which contain the LWS visual pigment. Conventional rods were not observed, although the MWS photoreceptor may be a "transmuted" rod. We also used stereology to measure the total number and topographic distribution of neurons in the ganglion cell layer of L. curtus, the olive sea snake (Aipysurus laevis), and the olive-headed sea snake (Disteira major). All species have a horizontal visual streak with specialized areas in the nasal and temporal retina. Both L. curtus and D. major also have a specialized area in the ventral retina, which may reflect differences in habitat usage and/or foraging behavior compared to A. laevis. Maximal spatial resolution was estimated at 1.1, 1.6, and 2.3 cycles deg⁻¹ in D. major, L. curtus, and A. laevis, respectively; the superior value for A. laevis may reflect its specialized crevice-foraging hunting technique.

  3. Membrane Guanylyl Cyclase Complexes Shape the Photoresponses of Retinal Rods and Cones

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    Xiao-Hong eWen

    2014-06-01

    Full Text Available In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP and the subsequent closure of cyclic nucleotide gated (CNG ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs for cGMP synthesis. Ten neuronal Ca2+ sensor proteins could potentially modulate their activities. The mouse is proving to be an effective model for characterizing the roles of individual components because its relative simplicity can be reduced further by genetic engineering. There are two types of guanylyl cyclase activating proteins (GCAPs and two types of GCs in mouse rods, whereas cones express one type of GCAP and one type of GC. Mutant mouse rods and cones bereft of both GCAPs have large, long lasting photon responses. Thus, GCAPs normally mediate negative feedback tied to the light-induced decline in intracellular Ca2+ that accelerates GC activity to curtail the growth and duration of the photon response. Rods from other mutant mice that express a single GCAP type reveal how the two GCAPs normally work together as a team. Because of its lower Ca2+ affinity, GCAP1 is the first responder that senses the initial decrease in Ca2+ following photon absorption and acts to limit response amplitude. GCAP2, with a higher Ca2+ affinity, is recruited later during the course of the photon response as Ca2+ levels continue to decline further. The main role of GCAP2 is to provide for a timely response recovery and it is particularly important after exposure to very bright light. The multiplicity of GC isozymes and GCAP homologs in the retinas of other vertebrates confers greater flexibility in shaping the photon responses in order to tune visual sensitivity, dynamic range and frequency response.

  4. Auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism

    Science.gov (United States)

    Cai, Weiming; Hu, Liwei; Hu, Xiangyang; Cui, Dayong; Cai, Weiming

    Gravitropism is the asymmetric growth or curvature of plant organs in response to gravistimulation. There is a complex signal transduction cascade which involved in the differential growth of plants in response to changes in the gravity vector. The role of auxin in gravitropism has been demonstrated by many experiments, but little is known regarding the molecular details of such effects. In our studies before, mediation of the gravitropic bending of soybean roots and rice leaf sheath bases by nitric oxide, cGMP and gibberellins, are induced by auxin. The asymmetrical distribution of nitric oxide, cGMP and gibberellins resulted from the asymmetrical synthesis of them in bending sites. In soybean roots, inhibitions of NO and cGMP synthesis reduced differential NO and cGMP accumulation respectively, which both of these effects can lead to the reduction of gravitropic bending. Gibberellin-induced OsXET, OsEXPA4 and OsRWC3 were also found involved in the gravitropic bending. These data indicated that auxin-induced nitric oxide, cGMP and gibberellins were involved in the gravitropism. More experiments need to prove the more detailed mechanism of them.

  5. Bax-induced apoptosis in Leber's congenital amaurosis: a dual role in rod and cone degeneration.

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    Séverine Hamann

    Full Text Available Pathogenesis in the Rpe65(-/- mouse model of Leber's congenital amaurosis (LCA is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65(-/- mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65, was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65(-/- mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65(-/-/Gnat1(-/- mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65(-/-/Bax(-/- mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65(-/- mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65(-/-/Bax(-/- mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice

  6. Replacement gene therapy with a human RPGRIP1 sequence slows photoreceptor degeneration in a murine model of Leber congenital amaurosis.

    Science.gov (United States)

    Pawlyk, Basil S; Bulgakov, Oleg V; Liu, Xiaoqing; Xu, Xiaoyun; Adamian, Michael; Sun, Xun; Khani, Shahrokh C; Berson, Eliot L; Sandberg, Michael A; Li, Tiansen

    2010-08-01

    RPGR-interacting protein-1 (RPGRIP1) is localized in the photoreceptor-connecting cilium, where it anchors the RPGR (retinitis pigmentosa GTPase regulator) protein, and its function is essential for photoreceptor maintenance. Genetic defect in RPGRIP1 is a known cause of Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration. We evaluated the efficacy of replacement gene therapy in a murine model of LCA carrying a targeted disruption of RPGRIP1. The replacement construct, packaged in an adeno-associated virus serotype 8 (AAV8) vector, used a rhodopsin kinase gene promoter to drive RPGRIP1 expression. Both promoter and transgene were of human origin. After subretinal delivery of the replacement gene in the mutant mice, human RPGRIP1 was expressed specifically in photoreceptors, localized correctly in the connecting cilia, and restored the normal localization of RPGR. Electroretinogram and histological examinations showed better preservation of rod and cone photoreceptor function and improved photoreceptor survival in the treated eyes. This study demonstrates the efficacy of human gene replacement therapy and validates a gene therapy design for future clinical trials in patients afflicted with this condition. Our results also have therapeutic implications for other forms of retinal degenerations attributable to a ciliary defect.

  7. Up-regulation of tumor necrosis factor superfamily genes in early phases of photoreceptor degeneration.

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    Sem Genini

    Full Text Available We used quantitative real-time PCR to examine the expression of 112 genes related to retinal function and/or belonging to known pro-apoptotic, cell survival, and autophagy pathways during photoreceptor degeneration in three early-onset canine models of human photoreceptor degeneration, rod cone dysplasia 1 (rcd1, X-linked progressive retinal atrophy 2 (xlpra2, and early retinal degeneration (erd, caused respectively, by mutations in PDE6B, RPGRORF15, and STK38L. Notably, we found that expression and timing of differentially expressed (DE genes correlated with the cell death kinetics. Gene expression profiles of rcd1 and xlpra2 were similar; however rcd1 was more severe as demonstrated by the results of the TUNEL and ONL thickness analyses, a greater number of genes that were DE, and the identification of altered expression that occurred at earlier time points. Both diseases differed from erd, where a smaller number of genes were DE. Our studies did not highlight the potential involvement of mitochondrial or autophagy pathways, but all three diseases were accompanied by the down-regulation of photoreceptor genes, and up-regulation of several genes that belong to the TNF superfamily, the extrinsic apoptotic pathway, and pro-survival pathways. These proteins were expressed by different retinal cells, including horizontal, amacrine, ON bipolar, and Müller cells, and suggest an interplay between the dying photoreceptors and inner retinal cells. Western blot and immunohistochemistry results supported the transcriptional regulation for selected proteins. This study highlights a potential role for signaling through the extrinsic apoptotic pathway in early cell death events and suggests that retinal cells other than photoreceptors might play a primary or bystander role in the degenerative process.

  8. Meckelin 3 is necessary for photoreceptor outer segment development in rat Meckel syndrome.

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    Sarika Tiwari

    Full Text Available Ciliopathies lead to multiorgan pathologies that include renal cysts, deafness, obesity and retinal degeneration. Retinal photoreceptors have connecting cilia joining the inner and outer segment that are responsible for transport of molecules to develop and maintain the outer segment process. The present study evaluated meckelin (MKS3 expression during outer segment genesis and determined the consequences of mutant meckelin on photoreceptor development and survival in Wistar polycystic kidney disease Wpk/Wpk rat using immunohistochemistry, analysis of cell death and electron microscopy. MKS3 was ubiquitously expressed throughout the retina at postnatal day 10 (P10 and P21. However, in the mature retina, MKS3 expression was restricted to photoreceptors and the retinal ganglion cell layer. At P10, both the wild type and homozygous Wpk mutant retina had all retinal cell types. In contrast, by P21, cells expressing rod- and cone-specific markers were fewer in number and expression of opsins appeared to be abnormally localized to the cell body. Cell death analyses were consistent with the disappearance of photoreceptor-specific markers and showed that the cells were undergoing caspase-dependent cell death. By electron microscopy, P10 photoreceptors showed rudimentary outer segments with an axoneme, but did not develop outer segment discs that were clearly present in the wild type counterpart. At p21 the mutant outer segments appeared much the same as the P10 mutant outer segments with only a short axoneme, while the wild-type controls had developed outer segments with many well-organized discs. We conclude that MKS3 is not important for formation of connecting cilium and rudimentary outer segments, but is critical for the maturation of outer segment processes.

  9. Essential Role of the Chaperonin CCT in Rod Outer Segment Biogenesis

    Science.gov (United States)

    Sinha, Satyabrata; Belcastro, Marycharmain; Datta, Poppy; Seo, Seongjin; Sokolov, Maxim

    2014-01-01

    Purpose. While some evidence suggests an essential role for the chaperonin containing t-complex protein 1 (CCT) in ciliogenesis, this function remains poorly understood mechanistically. We used transgenic mice, previously generated in our lab, and characterized by a genetically-induced suppression of CCT in rod photoreceptors as well as a malformation of the rod sensory cilia, the outer segments, to gain new insights into this underlying molecular mechanism. Methods. The CCT activity in rod photoreceptors of mice was suppressed by overexpressing the chaperonin inhibitor, phosducin-like protein short, and the ensuing changes of cellular morphology were analyzed by light and electron microscopy. Protein expression levels were studied by fluorescent microscopy and Western blotting. Results. Suppressing the chaperonin made the photoreceptors incompetent to build their outer segments. Specifically, the CCT-deficient rods appeared unable to expand the outer segment plasma membrane, and accommodate growth of this compartment. Seeking the molecular mechanisms underlying such a shortcoming, we found that the affected rods could not express normal levels of Bardet-Biedl Syndrome (BBS) proteins 2, 5, and 7 and, owing to that deficiency, were unable to assemble the BBSome, a multisubunit complex responsible for ciliary trafficking. A similar effect in response to the chaperonin suppression was also observed in cultured ciliated cells. Conclusions. Our data provide new evidence indicating the essential role of the chaperonin CCT in the biogenesis of vertebrate photoreceptor sensory cilia, and suggest that it may be due to the direct participation of the chaperonin in the posttranslational processing of selected BBS proteins and assembly of the BBSome. PMID:24854858

  10. Differential effects of nitric oxide on rod and cone pathways in carp retina

    Institute of Scientific and Technical Information of China (English)

    叶冰; 杜久林; 杨雄里

    1997-01-01

    The effects of nitric oxide (NO) on electroretinograms and light responses of horizontal cells intra-cellularly recorded from isolated, superfused carp retinas were studied. Sodium nitroprusside (SNP), an NO donor, suppressed scotopic b wave, while enhancing photopic b wave, and the effects could be blocked by hemoglobin, an NO chelator. Furthermore, following SNP application, light responses of rod horizontal cells were reduced in size and those of cone horizontal cells were increased. These results suggest that NO suppresses the activity of rod pathway, but enhances that of cone pathway in the outer retina. Moreover, the effects of methylene blue, an inhibitor of soluble guanylate cyclase, on rod and cone horizontal cells were just opposite to those of SNP, implying that the effects of NO may be mediated by cGMP.

  11. Synaptogenesis and synaptic protein localization in the postnatal development of rod bipolar cell dendrites in mouse retina.

    Science.gov (United States)

    Anastassov, Ivan A; Wang, Weiwei; Dunn, Felice A

    2017-05-25

    Retinal responses to photons originate in rod photoreceptors and are transmitted to the ganglion cell output of the retina through the primary rod bipolar pathway. At the first synapse of this pathway, input from multiple rods is pooled into individual rod bipolar cells. This architecture is called convergence. Convergence serves to improve sensitivity of rod vision when photons are sparse. Establishment of convergence depends on the development of a proper complement of dendritic tips and transduction proteins in rod bipolar cells. How the dendrites of rod bipolar cells develop and contact the appropriate number of rods is unknown. To answer this question we visualized individual rod bipolar cells in mouse retina during postnatal development and quantified the number of dendritic tips, as well as the expression of transduction proteins within dendrites. Our findings show that the number of dendritic tips in rod bipolar cells increases monotonically during development. The number of tips at P21, P30, and P82 exceeds the previously reported rod convergence ratios, and the majority of these tips are proximal to a presynaptic rod release site, suggesting more rods provide input to a rod bipolar cell. We also show that dendritic transduction cascade members mGluR6 and TRPM1 appear in tips with different timelines. These finding suggest that (a) rod bipolar cell dendrites elaborate without pruning during development, (b) the convergence ratio between rods and rod bipolar cells may be higher than previously reported, and (c) mGluR6 and TRPM1 are trafficked independently during development. © 2017 Wiley Periodicals, Inc.

  12. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP.

    Science.gov (United States)

    Morita, T; Perrella, M A; Lee, M E; Kourembanas, S

    1995-02-28

    Carbon monoxide (CO) is a product of the enzyme heme oxygenase (HO; EC 1.14.99.3). In vascular smooth muscle cells, exogenously administered CO increases cyclic guanosine 3',5'-monophosphate (cGMP), which is an important regulator of vessel tone. We report here that smooth muscle cells produce CO via HO and that it regulates cGMP levels in these cells. Hypoxia, which has profound effects on vessel tone, significantly increased the transcriptional rate of the HO-1 gene resulting in corresponding increases of its mRNA and HO enzymatic activity. In addition, under the same conditions, rat aortic and pulmonary artery smooth muscle cells accumulated high levels of cGMP following a similar time course to that of HO-1 production. The increased accumulation of cGMP in smooth muscle cells required the enzymatic activity of HO, since it was abolished by a specific HO inhibitor, tin protoporphyrin. In contrast, N omega-nitro-L-arginine, a potent inhibitor of nitric oxide (NO) synthesis, had no effect on cGMP produced by smooth muscle cells, indicating that NO is not responsible for the activation of guanylyl cyclase in this setting. Furthermore, conditioned medium from hypoxic smooth muscle cells stimulated cGMP production in recipient cells and this stimulation was completely inhibited by tin protoporphyrin or hemoglobin, an inhibitor of CO production and a scavenger of CO, respectively. This report shows that HO-1 is expressed by vascular smooth muscle cells and that its product, CO, may regulate vascular tone under physiologic and pathophysiologic (such as hypoxic) conditions.

  13. Receptors and cGMP signalling mechanism for E. coli enterotoxin in opossum kidney

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    Forte, L.R.; Krause, W.J.; Freeman, R.H. (Univ. of Missouri, Columbia (USA) Harry S. Truman Memorial Veterans Medical Center, Columbia, MO (USA))

    1988-11-01

    Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3{prime},5{prime}-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with {sup 125}I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments.

  14. Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

    Science.gov (United States)

    Lakowski, Jorn; Gonzalez-Cordero, Anai; West, Emma L; Han, Ya-Ting; Welby, Emily; Naeem, Arifa; Blackford, Samuel J I; Bainbridge, James W B; Pearson, Rachael A; Ali, Robin R; Sowden, Jane C

    2015-08-01

    Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(-) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application.

  15. Combinatorial Regulation of Photoreceptor Differentiation Factor, Neural Retina Leucine Zipper Gene Nrl, Revealed by in Vivo Promoter Analysis*

    Science.gov (United States)

    Kautzmann, Marie-Audrey I.; Kim, Douglas S.; Felder-Schmittbuhl, Marie-Paule; Swaroop, Anand

    2011-01-01

    Development and homeostasis require stringent spatiotemporal control of gene expression patterns that are established, to a large extent, by combinatorial action of transcription regulatory proteins. The bZIP transcription factor NRL (neural retina leucine zipper) is critical for rod versus cone photoreceptor cell fate choice during retinal development and acts as a molecular switch to produce rods from postmitotic precursors. Loss of Nrl in mouse leads to a cone-only retina, whereas ectopic expression of Nrl in photoreceptor precursors generates rods. To decipher the transcriptional regulatory mechanisms upstream of Nrl, we identified putative cis-control elements in the Nrl promoter/enhancer region by examining cross-species sequence conservation. Using in vivo transfection of promoter-reporter constructs into the mouse retina, we show that a 0.9-kb sequence upstream of the Nrl transcription initiation site is sufficient to drive reporter gene expression in photoreceptors. We further define a 0.3-kb sequence including a proximal promoter (cluster A1) and an enhancer (cluster B) that can direct rod-specific expression in vivo. Electrophoretic mobility shift assays using mouse retinal nuclear extracts, in combination with specific antibodies, demonstrate the binding of retinoid-related orphan nuclear receptor β (RORβ), cone rod homeobox, orthodenticle homolog 2, and cyclic AMP response element-binding protein to predicted consensus elements within clusters A and B. Our studies demonstrate Nrl as a direct transcriptional target of RORβ and suggest that combinatorial action of multiple regulatory factors modulates the expression of Nrl in developing and mature retina. PMID:21673114

  16. Combinatorial regulation of photoreceptor differentiation factor, neural retina leucine zipper gene NRL, revealed by in vivo promoter analysis.

    Science.gov (United States)

    Kautzmann, Marie-Audrey I; Kim, Douglas S; Felder-Schmittbuhl, Marie-Paule; Swaroop, Anand

    2011-08-12

    Development and homeostasis require stringent spatiotemporal control of gene expression patterns that are established, to a large extent, by combinatorial action of transcription regulatory proteins. The bZIP transcription factor NRL (neural retina leucine zipper) is critical for rod versus cone photoreceptor cell fate choice during retinal development and acts as a molecular switch to produce rods from postmitotic precursors. Loss of Nrl in mouse leads to a cone-only retina, whereas ectopic expression of Nrl in photoreceptor precursors generates rods. To decipher the transcriptional regulatory mechanisms upstream of Nrl, we identified putative cis-control elements in the Nrl promoter/enhancer region by examining cross-species sequence conservation. Using in vivo transfection of promoter-reporter constructs into the mouse retina, we show that a 0.9-kb sequence upstream of the Nrl transcription initiation site is sufficient to drive reporter gene expression in photoreceptors. We further define a 0.3-kb sequence including a proximal promoter (cluster A1) and an enhancer (cluster B) that can direct rod-specific expression in vivo. Electrophoretic mobility shift assays using mouse retinal nuclear extracts, in combination with specific antibodies, demonstrate the binding of retinoid-related orphan nuclear receptor β (RORβ), cone rod homeobox, orthodenticle homolog 2, and cyclic AMP response element-binding protein to predicted consensus elements within clusters A and B. Our studies demonstrate Nrl as a direct transcriptional target of RORβ and suggest that combinatorial action of multiple regulatory factors modulates the expression of Nrl in developing and mature retina.

  17. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP.

    OpenAIRE

    Morita, T.; Perrella, M A; Lee, M E; Kourembanas, S

    1995-01-01

    Carbon monoxide (CO) is a product of the enzyme heme oxygenase (HO; EC 1.14.99.3). In vascular smooth muscle cells, exogenously administered CO increases cyclic guanosine 3',5'-monophosphate (cGMP), which is an important regulator of vessel tone. We report here that smooth muscle cells produce CO via HO and that it regulates cGMP levels in these cells. Hypoxia, which has profound effects on vessel tone, significantly increased the transcriptional rate of the HO-1 gene resulting in correspondi...

  18. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

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    Viacheslav O. Nikolaev

    2013-04-01

    Full Text Available Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains.

  19. The co-chaperone and reductase ERdj5 facilitates rod opsin biogenesis and quality control.

    Science.gov (United States)

    Athanasiou, Dimitra; Bevilacqua, Dalila; Aguila, Monica; McCulley, Caroline; Kanuga, Naheed; Iwawaki, Takao; Chapple, J Paul; Cheetham, Michael E

    2014-12-15

    Mutations in rhodopsin, the light-sensitive protein of rod cells, are the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Many rod opsin mutations, such as P23H, lead to misfolding of rod opsin with detrimental effects on photoreceptor function and viability. Misfolded P23H rod opsin and other mutations in the intradiscal domain are characterized by the formation of an incorrect disulphide bond between C185 and C187, as opposed to the correct and highly conserved C110-C187 disulphide bond. Therefore, we tested the hypothesis that incorrect disulphide bond formation might be a factor that affects the biogenesis of rod opsin by studying wild-type (WT) or P23H rod opsin in combination with amino acid substitutions that prevent the formation of incorrect disulphide bonds involving C185. These mutants had altered traffic dynamics, suggesting a requirement for regulation of disulphide bond formation/reduction during rod opsin biogenesis. Here, we show that the BiP co-chaperone and reductase protein ERdj5 (DNAJC10) regulates this process. ERdj5 overexpression promoted the degradation, improved the endoplasmic reticulum mobility and prevented the aggregation of P23H rod opsin. ERdj5 reduction by shRNA delayed rod opsin degradation and promoted aggregation. The reductase and co-chaperone activity of ERdj5 were both required for these effects on P23H rod opsin. Furthermore, mutations in these functional domains acted as dominant negatives that affected WT rod opsin biogenesis. Collectively, these data identify ERdj5 as a member of the proteostasis network that regulates rod opsin biogenesis and supports a role for disulphide bond formation/reduction in rod opsin biogenesis and disease.

  20. Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

    Science.gov (United States)

    Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

    2002-11-01

    Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

  1. Photoreceptor Mediated Plant Growth Responses: Implications for Photoreceptor Engineering toward Improved Performance in Crops

    Directory of Open Access Journals (Sweden)

    Ophilia I. L. Mawphlang

    2017-07-01

    Full Text Available Rising temperatures during growing seasons coupled with altered precipitation rates presents a challenging task of improving crop productivity for overcoming such altered weather patterns and cater to a growing population. Light is a critical environmental factor that exerts a powerful influence on plant growth and development ranging from seed germination to flowering and fruiting. Higher plants utilize a suite of complex photoreceptor proteins to perceive surrounding red/far-red (phytochromes, blue/UV-A (cryptochromes, phototropins, ZTL/FKF1/LKP2, and UV-B light (UVR8. While genomic studies have also shown that light induces extensive reprogramming of gene expression patterns in plants, molecular genetic studies have shown that manipulation of one or more photoreceptors can result in modification of agronomically beneficial traits. Such information can assist researchers to engineer photoreceptors via genome editing technologies to alter expression or even sensitivity thresholds of native photoreceptors for targeting aspects of plant growth that can confer superior agronomic value to the engineered crops. Here we summarize the agronomically important plant growth processes influenced by photoreceptors in crop species, alongwith the functional interactions between different photoreceptors and phytohormones in regulating these responses. We also discuss the potential utility of synthetic biology approaches in photobiology for improving agronomically beneficial traits of crop plants by engineering designer photoreceptors.

  2. Preservation of cone photoreceptors after a rapid yet transient degeneration and remodeling in cone-only Nrl-/- mouse retina.

    Science.gov (United States)

    Roger, Jerome E; Ranganath, Keerthi; Zhao, Lian; Cojocaru, Radu I; Brooks, Matthew; Gotoh, Norimoto; Veleri, Shobi; Hiriyanna, Avinash; Rachel, Rivka A; Campos, Maria Mercedes; Fariss, Robert N; Wong, Wai T; Swaroop, Anand

    2012-01-11

    Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor neural retina leucine zipper (NRL). The loss of Nrl (Nrl(-/-)) in mice results in a retina with predominantly S-opsin-containing cones that exhibit molecular and functional characteristics of wild-type cones. Here, we report that Nrl(-/-) retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by 4 months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic electroretinogram. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl(-/-) mouse illustrates the long-term viability of cones in the absence of rods and retinal pigment epithelium defects in a rodless retina. We propose that Nrl(-/-) retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.

  3. THE STRUCTURE AND CONCENTRATION OF SOLIDS IN PHOTORECEPTOR CELLS STUDIED BY REFRACTOMETRY AND INTERFERENCE MICROSCOPY

    Science.gov (United States)

    Sidman, Richard L.

    1957-01-01

    Fragments of freshly obtained retinas of several vertebrate species were studied by refractometry, with reference to the structure of the rods and cones. The findings allowed a reassessment of previous descriptions based mainly on fixed material. The refractometric method was used also to measure the refractice indices and to calculate the concentrations of solids and water in the various cell segments. The main quantitative data were confirmed by interference microscopy. When examined by the method of refractometry the outer segments of freshly prepared retinal rods appear homogeneous. Within a few minutes a single eccentric longitudinal fiber appears, and transverse striations may develop. These changes are attributed to imbibition of water and swelling in structures normally too small for detection by light microscopy. The central "core" of outer segments and the chromophobic disc between outer and inner segments appear to be artifacts resulting from shrinkage during dehydration. The fresh outer segments of cones, and the inner segments of rods and cones also are described and illustrated. The volumes, refractive indices, concentrations of solids, and wet and dry weights of various segments of the photoreceptor cells were tabulated. Rod outer segments of the different species vary more than 100-fold in volume and mass but all have concentrations of solids of 40 to 43 per cent. Cone outer segments contain only about 30 per cent solids. The myoids, paraboloids, and ellipsoids of the inner segments likewise have characteristic refractive indices and concentrations of solids. Some of the limitations and particular virtues of refractometry as a method for quantitative analysis of living cells are discussed in comparison with more conventional biochemical techniques. Also the shapes and refractive indices of the various segments of photoreceptor cells are considered in relation to the absorption and transmission of light. The Stiles-Crawford effect can be accounted

  4. A short history of cGMP, guanylyl cyclases, and cGMP-dependent protein kinases.

    Science.gov (United States)

    Kots, Alexander Y; Martin, Emil; Sharina, Iraida G; Murad, Ferid

    2009-01-01

    Here, we review the early studies on cGMP, guanylyl cyclases, and cGMP-dependent protein kinases to facilitate understanding of development of this exciting but complex field of research encompassing pharmacology, biochemistry, physiology, and molecular biology of these important regulatory molecules.

  5. A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function

    Directory of Open Access Journals (Sweden)

    Aileen Balkow

    2015-08-01

    Conclusions: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.

  6. Plasma levels of cAMP, cGMP and CGRP in sildenafil-induced headache

    DEFF Research Database (Denmark)

    Kruuse, Christina Rostrup; Frandsen, E; Schifter, S;

    2004-01-01

    Sildenafil, a selective inhibitor of the cyclic guanosine monophosphate (cGMP) degrading phosphodiestrase 5 (PDE5), induced migraine without aura in 10 of 12 migraine patients and in healthy subjects it induced significantly more headache than placebo. The aim of the present study was to determin...... an important role of these signalling molecules, the present study questions whether cAMP and cGMP in peripheral blood can be used for monitoring pathophysiological events in headache and migraine mechanisms.......Sildenafil, a selective inhibitor of the cyclic guanosine monophosphate (cGMP) degrading phosphodiestrase 5 (PDE5), induced migraine without aura in 10 of 12 migraine patients and in healthy subjects it induced significantly more headache than placebo. The aim of the present study was to determine...... whether the pain-inducing effects of sildenafil would be reflected in plasma levels of important signalling molecules in migraine: cGMP, cyclic adenosine monophosphate (cAMP) and calcitonin gene-related peptide (CGRP). Ten healthy subjects (four women, six men) and 12 patients (12 women) suffering from...

  7. HCN1 Channels Enhance Rod System Responsivity in the Retina under Conditions of Light Exposure.

    Directory of Open Access Journals (Sweden)

    Vithiyanjali Sothilingam

    Full Text Available Vision originates in rods and cones at the outer retina. Already at these early stages, diverse processing schemes shape and enhance image information to permit perception over a wide range of lighting conditions. In this work, we address the role of hyperpolarization-activated and cyclic nucleotide-gated channels 1 (HCN1 in rod photoreceptors for the enhancement of rod system responsivity under conditions of light exposure.To isolate HCN1 channel actions in rod system responses, we generated double mutant mice by crossbreeding Hcn1-/- mice with Cnga3-/- mice in which cones are non-functional. Retinal function in the resulting Hcn1-/- Cnga3-/- animals was followed by means of electroretinography (ERG up to the age of four month. Retinal imaging via scanning laser ophthalmoscopy (SLO and optical coherence tomography (OCT was also performed to exclude potential morphological alterations.This study on Hcn1-/- Cnga3-/- mutant mice complements our previous work on HCN1 channel function in the retina. We show here in a functional rod-only setting that rod responses following bright light exposure terminate without the counteraction of HCN channels much later than normal. The resulting sustained signal elevation does saturate the retinal network due to an intensity-dependent reduction in the dynamic range. In addition, the lack of rapid adaptational feedback modulation of rod photoreceptor output via HCN1 in this double mutant limits the ability to follow repetitive (flicker stimuli, particularly under mesopic conditions.This work corroborates the hypothesis that, in the absence of HCN1-mediated feedback, the amplitude of rod signals remains at high levels for a prolonged period of time, leading to saturation of the retinal pathways. Our results demonstrate the importance of HCN1 channels for regular vision.

  8. LINC complexes mediate the positioning of cone photoreceptor nuclei in mouse retina.

    Directory of Open Access Journals (Sweden)

    David Razafsky

    Full Text Available It has long been observed that many neuronal types position their nuclei within restricted cytoplasmic boundaries. A striking example is the apical localization of cone photoreceptors nuclei at the outer edge of the outer nuclear layer of mammalian retinas. Yet, little is known about how such nuclear spatial confinement is achieved and further maintained. Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes consist of evolutionary-conserved macromolecular assemblies that span the nuclear envelope to connect the nucleus with the peripheral cytoskeleton. Here, we applied a new transgenic strategy to disrupt LINC complexes either in cones or rods. In adult cones, we observed a drastic nuclear mislocalization on the basal side of the ONL that affected cone terminals overall architecture. We further provide evidence that this phenotype may stem from the inability of cone precursor nuclei to migrate towards the apical side of the outer nuclear layer during early postnatal retinal development. By contrast, disruption of LINC complexes within rod photoreceptors, whose nuclei are scattered across the outer nuclear layer, had no effect on the positioning of their nuclei thereby emphasizing differential requirements for LINC complexes by different neuronal types. We further show that Sun1, a component of LINC complexes, but not A-type lamins, which interact with LINC complexes at the nuclear envelope, participate in cone nuclei positioning. This study provides key mechanistic aspects underlying the well-known spatial confinement of cone nuclei as well as a new mouse model to evaluate the pathological relevance of nuclear mispositioning.

  9. Proteomics of photoreceptor outer segments identifies a subset of SNARE and Rab proteins implicated in membrane vesicle trafficking and fusion.

    Science.gov (United States)

    Kwok, Michael C M; Holopainen, Juha M; Molday, Laurie L; Foster, Leonard J; Molday, Robert S

    2008-06-01

    The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.

  10. Beta-ionone activates and bleaches visual pigment in salamander photoreceptors.

    Science.gov (United States)

    Isayama, Tomoki; McCabe England, S L; Crouch, R K; Zimmerman, A L; Makino, C L

    2009-01-01

    Vision begins with photoisomerization of 11-cis retinal to the all-trans conformation within the chromophore-binding pocket of opsin, leading to activation of a biochemical cascade. Release of all-trans retinal from the binding pocket curtails but does not fully quench the ability of opsin to activate transducin. All-trans retinal and some other analogs, such as beta-ionone, enhance opsin's activity, presumably on binding the empty chromophore-binding pocket. By recording from isolated salamander photoreceptors and from patches of rod outer segment membrane, we now show that high concentrations of beta-ionone suppressed circulating current in dark-adapted green-sensitive rods by inhibiting the cyclic nucleotide-gated channels. There were also decreases in circulating current and flash sensitivity, and accelerated flash response kinetics in dark-adapted blue-sensitive (BS) rods and cones, and in ultraviolet-sensitive cones, at concentrations too low to inhibit the channels. These effects persisted in BS rods even after incubation with 9-cis retinal to ensure complete regeneration of their visual pigment. After long exposures to high concentrations of beta-ionone, recovery was incomplete unless 9-cis retinal was given, indicating that visual pigment had been bleached. Therefore, we propose that beta-ionone activates and bleaches some types of visual pigments, mimicking the effects of light.

  11. Transcriptional Regulation of Neural Retina Leucine Zipper (Nrl), a Photoreceptor Cell Fate Determinant*

    Science.gov (United States)

    Montana, Cynthia L.; Lawrence, Karen A.; Williams, Natecia L.; Tran, Nicholas M.; Peng, Guang-Hua; Chen, Shiming; Corbo, Joseph C.

    2011-01-01

    The transcription factor neural retina leucine zipper (Nrl) is a critical determinant of rod photoreceptor cell fate and a key regulator of rod differentiation. Nrl−/− rod precursors fail to turn on rod genes and instead differentiate as cones. Furthermore, NRL mutations in humans cause retinitis pigmentosa. Despite the developmental and clinical significance of this gene, little is known about the transcriptional regulation of Nrl itself. In this study, we sought to define the cis- and trans-acting factors responsible for initiation and maintenance of Nrl transcription in the mouse retina. Utilizing a quantitative mouse retinal explant electroporation assay, we discovered a phylogenetically conserved, 30-base pair region immediately upstream of the transcription start site that is required for Nrl promoter activity. This region contains binding sites for the retinal transcription factors CRX, OTX2, and RORβ, and point mutations in these sites completely abolish promoter activity in living retinas. Gel-shift experiments show that CRX, OTX2, and RORβ can bind to the critical region in vitro, whereas ChIP experiments demonstrate binding of CRX and OTX2 to the critical region in vivo. Thus, our results indicate that CRX, OTX2, and RORβ directly regulate Nrl transcription by binding to critical sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by OTX2 and RORβ and later maintained at high levels by CRX and RORβ. PMID:21865162

  12. Transcriptional regulation of neural retina leucine zipper (Nrl), a photoreceptor cell fate determinant.

    Science.gov (United States)

    Montana, Cynthia L; Lawrence, Karen A; Williams, Natecia L; Tran, Nicholas M; Peng, Guang-Hua; Chen, Shiming; Corbo, Joseph C

    2011-10-21

    The transcription factor neural retina leucine zipper (Nrl) is a critical determinant of rod photoreceptor cell fate and a key regulator of rod differentiation. Nrl(-/-) rod precursors fail to turn on rod genes and instead differentiate as cones. Furthermore, NRL mutations in humans cause retinitis pigmentosa. Despite the developmental and clinical significance of this gene, little is known about the transcriptional regulation of Nrl itself. In this study, we sought to define the cis- and trans-acting factors responsible for initiation and maintenance of Nrl transcription in the mouse retina. Utilizing a quantitative mouse retinal explant electroporation assay, we discovered a phylogenetically conserved, 30-base pair region immediately upstream of the transcription start site that is required for Nrl promoter activity. This region contains binding sites for the retinal transcription factors CRX, OTX2, and RORβ, and point mutations in these sites completely abolish promoter activity in living retinas. Gel-shift experiments show that CRX, OTX2, and RORβ can bind to the critical region in vitro, whereas ChIP experiments demonstrate binding of CRX and OTX2 to the critical region in vivo. Thus, our results indicate that CRX, OTX2, and RORβ directly regulate Nrl transcription by binding to critical sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by OTX2 and RORβ and later maintained at high levels by CRX and RORβ.

  13. A study of the human rod and cone electroretinogram a-wave component

    Science.gov (United States)

    Barraco, R.; Persano Adorno, D.; Bellomonte, L.; Brai, M.

    2009-03-01

    The study of the electrical response of the retina to a luminous stimulus is one of the main fields of research in ocular electrophysiology. The features of the first component (a-wave) of the retinal response reflect the functional integrity of the two populations of photoreceptors: rods and cones. We fit the a-wave for pathological subjects with functions that account for possible mechanisms governing the kinetics of the photoreceptors. The paper extends a previous analysis, carried out for normal subjects, in which both populations are active, to patients affected by two particular diseases that reduce the working populations to only one. The pathologies investigated are Achromatopsia, a cone disease, and Congenital Stationary Night Blindness, a rod problem. We present evidence that the analysis of a pathological a-wave can be employed to quantitatively measure either cone or rod activities and to test hypotheses about their responses. The results show that the photoreceptoral responses differ in the two cases and functions implying a different number of photocascade stages are necessary to achieve a correct modeling of the early phototransduction process. Numerical values of the parameters characterizing the best-fit functions are given and discussed.

  14. Developmental dynamics of cone photoreceptors in the eel

    Directory of Open Access Journals (Sweden)

    Semo Ma'ayan

    2009-12-01

    Full Text Available Abstract Background Many fish alter their expressed visual pigments during development. The number of retinal opsins expressed and their type is normally related to the environment in which they live. Eels are known to change the expression of their rod opsins as they mature, but might they also change the expression of their cone opsins? Results The Rh2 and Sws2 opsin sequences from the European Eel were isolated, sequenced and expressed in vitro for an accurate measurement of their λmax values. In situ hybridisation revealed that glass eels express only rh2 opsin in their cone photoreceptors, while larger yellow eels continue to express rh2 opsin in the majority of their cones, but also have Conclusions Larger yellow and silver European eels express two different cone opsins, rh2 and sws2. This work demonstrates that only the Rh2 cone opsin is present in younger fish (smaller yellow and glass, the sws2 opsin being expressed additionally only by older fish and only in

  15. Small Molecules that Protect Mitochondrial Function from Metabolic Stress Decelerate Loss of Photoreceptor Cells in Murine Retinal Degeneration Models.

    Science.gov (United States)

    Beeson, Craig; Lindsey, Chris; Nasarre, Cecile; Bandyopadhyay, Mausumi; Perron, Nathan; Rohrer, Bärbel

    2016-01-01

    One feature common to many of the pathways implicated in retinal degeneration is increased metabolic stress leading to impaired mitochondrial function. We found that exposure of cells to calcium ionophores or oxidants as metabolic stressors diminish maximal mitochondrial capacity. A library of 50,000 structurally diverse "drug-like" molecules was screened for protection against loss of calcium-induced loss of mitochondrial capacity in 661W rod-derived cells and C6 glioblastomas. Initial protective hits were then tested for protection against IBMX-induced loss of mitochondrial capacity as measured via respirometry. Molecules that protected mitochondria were then evaluated for protection of rod photoreceptor cells in retinal explants from rd1 mice. Two of the molecules attenuated loss of photoreceptor cells in the rd1 model. In the 661W cells, exposure to calcium ionophore or tert-butylhydroperoxide caused mitochondrial fragmentation that was blocked with the both compounds. Our studies have identified molecules that protect mitochondria and attenuate loss of photoreceptors in models of retinal degeneration suggesting that they could be good leads for development of therapeutic drugs for treatment of a wide variety of retinal dystrophies.

  16. A single valine residue plays an essential role in peripherin/rds targeting to photoreceptor outer segments.

    Science.gov (United States)

    Salinas, Raquel Y; Baker, Sheila A; Gospe, Sidney M; Arshavsky, Vadim Y

    2013-01-01

    Peripherin/retinal degeneration slow (rds) is an integral membrane protein specifically localized to the light-sensing organelle of the photoreceptor cell, the outer segment. Within the outer segment, peripherin is found at the edges of photoreceptor discs, where it plays a critical role in disc morphogenesis and maintenance. Peripherin loss or mutations are often associated with severe forms of visual impairments. Like all other resident outer segment proteins, peripherin is synthesized in the photoreceptor cell body and subsequently transported to the outer segment. In an effort to further examine peripherin's delivery to outer segments, we undertook a careful examination of its targeting sequence. Using a fluorescently labeled reporter expressed in the rods of transgenic tadpoles, we narrowed peripherin's targeting sequence to ten amino acids within its C-terminal tail. This small stretch of amino acid residues is both necessary and sufficient for outer segment targeting. We also conducted alanine scanning of all residues within this sequence and found that only a single residue, valine at position 332, is essential for outer segment targeting. This valine is conserved in all species and its mutation is sufficient to completely abrogate the targeting of full-length peripherin in mouse rods.

  17. MAS NMR study of the photoreceptor phytochrome

    NARCIS (Netherlands)

    Rohmer, Thierry

    2009-01-01

    Plants, algae and bacteria respond to light in various manners. The effect of light on the growth of plants is called photomorphogenesis and is regulated by the photoreceptor protein named phytochrome. Phytochrome is formed in the dark in its inactive red-absorbing (Pr) state and transformed upon ab

  18. Calcium homeostasis in fly photoreceptor cells

    NARCIS (Netherlands)

    Oberwinkler, J

    2002-01-01

    In fly photoreceptor cells, two processes dominate the Ca2+ homeostasis: light-induced Ca2+ influx through members of the TRP family of ion channels, and Ca2+ extrusion by Na+/Ca2+ exchange.Ca2+ release from intracellular stores is quantitatively insignificant. Both, the light-activated channels and

  19. Overexpressed or intraperitoneally injected human transferrin prevents photoreceptor degeneration in rd10 mice.

    Science.gov (United States)

    Picard, Emilie; Jonet, Laurent; Sergeant, Claire; Vesvres, Marie-Hélène; Behar-Cohen, Francine; Courtois, Yves; Jeanny, Jean-Claude

    2010-12-08

    Retinal degeneration has been associated with iron accumulation in age-related macular degeneration (AMD), and in several rodent models that had one or several iron regulating protein impairments. We investigated the iron concentration and the protective role of human transferrin (hTf) in rd10 mice, a model of retinal degeneration. The proton-induced X-ray emission (PIXE) method was used to quantify iron in rd10 mice 2, 3, and 4 weeks after birth. We generated mice with the β-phosphodiesterase mutation and hTf expression by crossbreeding rd10 mice with TghTf mice (rd10/hTf mice). The photoreceptor loss and apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling in 3-week-old rd10/hTf mice and compared with 3-week-old rd10 mice. The neuroprotective effect of hTf was analyzed in 5-day-old rd10 mice treated by intraperitoneal administration with hTf for up to 25 days. The retinal hTf concentrations and the thickness of the outer nuclear layer were quantified in all treated mice at 25 days postnatally. PIXE analysis demonstrated an age-dependent iron accumulation in the photoreceptors of rd10 mice. The rd10/hTf mice had the rd10 mutation, expressed high levels of hTf, and showed a significant decrease in photoreceptor death. In addition, rd10 mice intraperitoneally treated with hTf resulted in the retinal presence of hTf and a dose-dependent reduction in photoreceptor degeneration. Our results suggest that iron accumulation in the retinas of rd10 mutant mice is associated with photoreceptor degeneration. For the first time, the enhanced survival of cones and rods in the retina of this model has been demonstrated through overexpression or systemic administration of hTf. This study highlights the therapeutic potential of Tf to inhibit iron-induced photoreceptor cell death observed in degenerative diseases such as retinitis pigmentosa and age-related macular degeneration.

  20. Morphological characterization and topographic analysis of multiple photoreceptor types in the retinae of mesopelagic hatchetfishes with tubular eyes

    Directory of Open Access Journals (Sweden)

    Lauren Michelle Biagioni

    2016-03-01

    Full Text Available Marine hatchetfishes, Argyropelecus spp., are one of the 14 genera of mesopelagic teleosts, which possess tubular eyes. The tubular eyes are positioned dorsally on the head and consist of a main retina, which subtends a large dorsal binocular field, and an accessory retina, which subtends the lateral monocular visual field. The topographic distribution of photoreceptors in the retina of Argyropelecus sladeni, A. affinis and A. aculeatus was determined using a random, unbiased and systematic stereological approach, which consistently revealed a region of high density (area centralis in the central region of the main retina (up to a peak of 96,000 receptors per mm2 and a relatively homogeneous density of photoreceptors in the accessory retina (of approximately 20,000 receptors per mm2. The position of the area centralis in the main retina indicates this retinal region subserves greater spatial resolution in the centre of the dorsal binocular visual field. Light microscopy and transmission electron microscopy also revealed the presence of multiple photoreceptor types (two rod-like and one cone-like based on the size and shape of the inner and outer segments and ultrastructural differences in the ellipsoidal region. The presence of multiple photoreceptor types in these tubular-eyed, mesopelagic hatchetfishes may reflect the need for the visual system to function under different lighting conditions during vertical migratory behavior, especially given their unique dorsally-facing eyes.

  1. Filtration behavior of casein glycomacropeptide (CGMP) in an enzymatic membrane reactor: fouling control by membrane selection and threshold flux operation

    DEFF Research Database (Denmark)

    Luo, Jianquan; Morthensen, Sofie Thage; Meyer, Anne S.

    2014-01-01

    . In this study, the filtration performance and fouling behavior during ultrafiltration (UF) of CGMP for the enzymatic production of 3′-sialyllactose were investigated. A 5kDa regenerated cellulose membrane with high anti-fouling performance, could retain CGMP well, permeate 3′-sialyllactose, and was found...... concentration on the threshold flux were studied based on the resistance-in-series model. Higher hydrophilicity of the membrane, elevated pH and agitation, and lower CGMP concentration were found to increase the threshold flux and decrease membrane fouling....

  2. IL-4 induces cAMP and cGMP in human monocytic cells

    Directory of Open Access Journals (Sweden)

    B. Dugas

    1995-01-01

    Full Text Available Human monocytes, preincubated with IFN-γ respond to IL-4 by a cGMP increase through activation of an inducible NO synthase. Here, IL-4 was found to induce an accumulation of cGMP (1 – 3 min and cAMP (20 – 25 min in unstimulated monocytes. This was impaired with NOS inhibitors, but also with EGTA and calcium/calmodulin inhibitors. These results suggest that: (1 IL-4 may stimulate different NOS isoforms in resting and IFN-γ activated monocytes, and (2 cAMP accumulation may be partially dependent on the NO pathway. By RT-PCR, a type III constitutive NOS mRNA was detected in U937 monocytic cells. IL-4 also increased the [Ca2+]i in these cells. Different NOS may thus be expressed in monocytic cells depending on their differentiation and the signals they receive.

  3. Gene therapy with a promoter targeting both rods and cones rescues retinal degeneration caused by AIPL1 mutations.

    Science.gov (United States)

    Sun, X; Pawlyk, B; Xu, X; Liu, X; Bulgakov, O V; Adamian, M; Sandberg, M A; Khani, S C; Tan, M-H; Smith, A J; Ali, R R; Li, T

    2010-01-01

    Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is required for the biosynthesis of photoreceptor phosphodiesterase (PDE). Gene defects in AIPL1 cause a heterogeneous set of conditions ranging from Leber's congenital amaurosis (LCA), the severest form of early-onset retinal degeneration, to milder forms such as retinitis pigmentosa (RP) and cone-rod dystrophy. In mice, null and hypomorphic alleles cause retinal degeneration similar to human LCA and RP, respectively. Thus these mouse models represent two ends of the disease spectrum associated with AIPL1 gene defects in humans. We evaluated whether adeno-associated virus (AAV)-mediated gene replacement therapy in these models could restore PDE biosynthesis in rods and cones and thereby improve photoreceptor survival. We validated the efficacy of human AIPL1 (isoform 1) replacement gene controlled by a promoter derived from the human rhodopsin kinase (RK) gene, which is active in both rods and cones. We found substantial and long-term rescue of the disease phenotype as a result of transgene expression. This is the first gene therapy study in which both rods and cones were targeted successfully with a single photoreceptor-specific promoter. We propose that the vector and construct design used in this study could serve as a prototype for a human clinical trial.

  4. A neuroanatomical and physiological study of the non-image forming visual system of the cone-rod homeobox gene (Crx) knock out mouse

    DEFF Research Database (Denmark)

    Rovsing, Louise; Rath, Martin F; Lund-Andersen, Casper

    2010-01-01

    The anatomy and physiology of the non-image forming visual system was investigated in a visually blind cone-rod homeobox gene (Crx) knock-out mouse (Crx(-)(/)(-)), which lacks the outer segments of the photoreceptors. We show that the suprachiasmatic nuclei (SCN) in the Crx(-/-) mouse exhibit...

  5. From bedside to bench--meeting report of the 7th International Conference on cGMP "cGMP: generators, effectors and therapeutic implications" in Trier, Germany, from June 19th to 21st 2015.

    Science.gov (United States)

    Friebe, Andreas; Sandner, Peter; Seifert, Roland

    2015-12-01

    During the past decade, our knowledge on the physiology, pathophysiology, basic pharmacology, and clinical pharmacology of the second messenger (cGMP) has increased tremendously. It is now well-established that cGMP, generated by soluble and particulate guanylate cyclases, is highly compartmentalized in cells and regulates numerous body functions. New cGMP-regulated physiological functions include meiosis and temperature perception. cGMP is involved in the genesis of numerous pathologies including cardiovascular, pulmonary, endocrine, metabolic, neuropsychiatric, eye, and tumor diseases. Several new clinical uses of stimulators and activators of soluble guanylate cyclase and of phosphodiesterase inhibitors such as heart failure, kidney failure, cognitive disorders, obesity bronchial asthma, and osteoporosis are emerging. The combination of neprilysin inhibitors-enhancing stimulation of the particulate guanylate cyclase pathway by preventing natriuretic peptide degradation-with angiotensin AT1 receptor antagonists constitutes a novel promising strategy for heart failure treatment. The role of oxidative stress in cGMP signaling, application of cGMP sensors, and gene therapy for degenerative eye diseases are emerging topics. It is anticipated that cGMP research will further prosper over the next years and reach out into more and more basic and clinical disciplines.

  6. Morphoelastic rods. Part I: A single growing elastic rod

    KAUST Repository

    Moulton, D.E.

    2013-02-01

    A theory for the dynamics and statics of growing elastic rods is presented. First, a single growing rod is considered and the formalism of three-dimensional multiplicative decomposition of morphoelasticity is used to describe the bulk growth of Kirchhoff elastic rods. Possible constitutive laws for growth are discussed and analysed. Second, a rod constrained or glued to a rigid substrate is considered, with the mismatch between the attachment site and the growing rod inducing stress. This stress can eventually lead to instability, bifurcation, and buckling. © 2012 Elsevier Ltd. All rights reserved.

  7. Learning with Rods: One Account.

    Science.gov (United States)

    Cherry, Donald Esha

    This paper discusses one English as a Second Language (ESL) teacher's attempts to use cuisenaire rods as a language learning tool. Cuisenaire rods (sometimes called algebricks) vary in size from 1 x 1 x 10 centimeter sticks to 1 x 1 x 1 centimeter cubes, with each of the 10 sizes a different color. Although such rods have been used to teach…

  8. Establishing a cGMP pancreatic islet processing facility: the first experience in Iran.

    Science.gov (United States)

    Larijani, Bagher; Arjmand, Babak; Amoli, Mahsa M; Ao, Ziliang; Jafarian, Ali; Mahdavi-Mazdah, Mitra; Ghanaati, Hossein; Baradar-Jalili, Reza; Sharghi, Sasan; Norouzi-Javidan, Abbas; Aghayan, Hamid Reza

    2012-12-01

    It has been predicted that one of the greatest increase in prevalence of diabetes will happen in the Middle East bear in the next decades. The aim of standard therapeutic strategies for diabetes is better control of complications. In contrast, some new strategies like cell and gene therapy have aimed to cure the disease. In recent years, significant progress has occurred in beta-cell replacement therapies with a progressive improvement of short-term and long term outcomes. In year 2005, considering the impact of the disease in Iran and the promising results of the Edmonton protocol, the funding for establishing a current Good Manufacturing Practice (cGMP) islet processing facility by Endocrinology and Metabolism Research Center was approved by Tehran University of Medical Sciences. Several islet isolations were performed following establishment of cGMP facility and recruitment of all required equipments for process validation and experimental purpose. Finally the first successful clinical islet isolation and transplantation was performed in September 2010. In spite of a high cost of the procedure it is considered beneficial and may prevent long term complications and the costs associated with secondary cares. In this article we will briefly describe our experience in setting up a cGMP islet processing facility which can provide valuable information for regional countries interested to establish similar facilities.

  9. The role of cGMP hydrolysing phosphodiesterases 1 and 5 in cerebral artery dilatation

    DEFF Research Database (Denmark)

    Kruuse, C; Rybalkin, S D; Khurana, T S;

    2001-01-01

    The aim was to investigate the presence and activity of cGMP hydrolysing phosphodiesterases in guinea pig basilar arteries and the effect of selective and non-selective phosphodiesterase inhibitors on cerebral artery dilatation involving the nitric oxide (NO)-guanosine cyclic 3'5-monophosphate (c...... by cGMP-independent mechanisms. Targeting the phosphodiesterases present in cerebral arteries, with selective inhibitors or activators of phosphodiesterase, may be a possible new way of treating cerebrovascular disease.......The aim was to investigate the presence and activity of cGMP hydrolysing phosphodiesterases in guinea pig basilar arteries and the effect of selective and non-selective phosphodiesterase inhibitors on cerebral artery dilatation involving the nitric oxide (NO)-guanosine cyclic 3'5-monophosphate (c......GMP) pathway. Immunoreactivity to phosphodiesterases 1A, 1B and 5, but not phosphodiesterase 1C was found in fractions of homogenised cerebral arteries eluted by high-pressure liquid chromatography (HPLC). Both the phosphodiesterase 1 inhibitor 8-methoxymethyl-1-methyl-3-(2methylpropyl)-xanthine (8-MM...

  10. A model for open-close control of cation channels in the plasma membrane of retinal rod outer segments.

    Science.gov (United States)

    Ichikawa, K

    1989-06-01

    A model for open-close control of cation channels in the plasma membrane of retinal rod outer segments is presented. A channel is assumed to open when 3 cGMP molecules bind to it and close as soon as one of the 3 cGMP molecules is released from it. The calcium ion (divalent cation) is a modulator of the channel conductance. The channel conductance is low when Ca2+ binds to it, while it is high when it is free from Ca2+. From the above assumptions, the reaction scheme of channels with cGMP and Ca2+ is created and the fraction of channels in the open and closed states was calculated using equations for this scheme. The kinetic constants used in the model are estimated from the experimental results of many studies and from the theories. From this estimation, it was found that at the physiological concentrations of intracellular and extracellular Ca2+, almost all channels are bound with Ca2+ and are in the low conductance state. The present model accounts for the reported dose(cGMP)-response(membrane current or conductance) relationship, where the Hill coefficient decreases as the cGMP concentration increases. The dark-level cGMP concentration of 8.13 microM is estimated from the model. This is in good agreement with the reported values. Moreover, the model predicts the invariance of current noise at relatively low Ca2+ concentrations when the cGMP concentration is raised from the dark level to a saturation level. The dynamic properties (opening and closing actions) of the channels in the present model are also in good agreement with the reported observations. The burst mode opening and closing of a channel is predicted by the present model, and it was found that the number of openings in a burst is controlled by the forward and backward rate constants between a channel protein and cGMP molecules. The simulated waveform of a single channel is similar to the reported observations.

  11. Insect photoreceptor adaptations to night vision.

    Science.gov (United States)

    Honkanen, Anna; Immonen, Esa-Ville; Salmela, Iikka; Heimonen, Kyösti; Weckström, Matti

    2017-04-05

    Night vision is ultimately about extracting information from a noisy visual input. Several species of nocturnal insects exhibit complex visually guided behaviour in conditions where most animals are practically blind. The compound eyes of nocturnal insects produce strong responses to single photons and process them into meaningful neural signals, which are amplified by specialized neuroanatomical structures. While a lot is known about the light responses and the anatomical structures that promote pooling of responses to increase sensitivity, there is still a dearth of knowledge on the physiology of night vision. Retinal photoreceptors form the first bottleneck for the transfer of visual information. In this review, we cover the basics of what is known about physiological adaptations of insect photoreceptors for low-light vision. We will also discuss major enigmas of some of the functional properties of nocturnal photoreceptors, and describe recent advances in methodologies that may help to solve them and broaden the field of insect vision research to new model animals.This article is part of the themed issue 'Vision in dim light'.

  12. Safety rod latch inspection

    Energy Technology Data Exchange (ETDEWEB)

    Leader, D.R.

    1992-02-01

    During an attempt to raise control rods from the 100 K reactor in December, one rod could not be withdrawn. Subsequent investigation revealed that a small button'' in the latch mechanism had broken off of the lock plunger'' and was wedged in a position that prevented rod withdrawal. Concern that this failure may have resulted from corrosion or some other metallurgical problem resulted in a request that SRL examine six typical latch mechanisms from the 100 L reactor by use of radiography and metallography. During the examination of the L-Area latches, a failed latch mechanism from the 100 K reactor was added to the investigation. Fourteen latches that had a history of problems were removed from K-Area and sent to SRL for inclusion in this study the week after the original seven assemblies were examined, bringing the total of latch assemblies discussed in this report to twenty one. Results of the examination of the K-Area latch that initiated this study is not included in this report.

  13. Safety rod latch inspection

    Energy Technology Data Exchange (ETDEWEB)

    Leader, D.R.

    1992-02-01

    During an attempt to raise control rods from the 100 K reactor in December, one rod could not be withdrawn. Subsequent investigation revealed that a small ``button`` in the latch mechanism had broken off of the ``lock plunger`` and was wedged in a position that prevented rod withdrawal. Concern that this failure may have resulted from corrosion or some other metallurgical problem resulted in a request that SRL examine six typical latch mechanisms from the 100 L reactor by use of radiography and metallography. During the examination of the L-Area latches, a failed latch mechanism from the 100 K reactor was added to the investigation. Fourteen latches that had a history of problems were removed from K-Area and sent to SRL for inclusion in this study the week after the original seven assemblies were examined, bringing the total of latch assemblies discussed in this report to twenty one. Results of the examination of the K-Area latch that initiated this study is not included in this report.

  14. Hypoxia increases the yield of photoreceptors differentiating from mouse embryonic stem cells and improves the modeling of retinogenesis in vitro.

    Science.gov (United States)

    Garita-Hernández, Marcela; Diaz-Corrales, Francisco; Lukovic, Dunja; González-Guede, Irene; Diez-Lloret, Andrea; Valdés-Sánchez, M Lourdes; Massalini, Simone; Erceg, Slaven; Bhattacharya, Shomi S

    2013-05-01

    Retinitis pigmentosa (RP), a genetically heterogeneous group of diseases together with age-related macular degeneration (AMD), are the leading causes of permanent blindness and are characterized by the progressive dysfunction and death of the light sensing photoreceptors of the retina. Due to the limited regeneration capacity of the mammalian retina, the scientific community has invested significantly in trying to obtain retinal progenitor cells from embryonic stem cells (ESC). These represent an unlimited source of retinal cells, but it has not yet been possible to achieve specific populations, such as photoreceptors, efficiently enough to allow them to be used safely in the future as cell therapy of RP or AMD. In this study, we generated a high yield of photoreceptors from directed differentiation of mouse ESC (mESC) by recapitulating crucial phases of retinal development. We present a new protocol of differentiation, involving hypoxia and taking into account extrinsic and intrinsic cues. These include niche-specific conditions as well as the manipulation of the signaling pathways involved in retinal development. Our results show that hypoxia promotes and improves the differentiation of mESC toward photoreceptors. Different populations of retinal cells are increased in number under the hypoxic conditions applied, such as Crx-positive cells, S-Opsin-positive cells, and double positive cells for Rhodopsin and Recoverin, as shown by immunofluorescence analysis. For the first time, this manuscript reports the high efficiency of differentiation in vivo and the expression of mature rod photoreceptor markers in a large number of differentiated cells, transplanted in the subretinal space of wild-type mice.

  15. Investigating photoreceptor densities, potential visual acuity, and cone mosaics of shallow water, temperate fish species.

    Science.gov (United States)

    Hunt, D E; Rawlinson, N J F; Thomas, G A; Cobcroft, J M

    2015-06-01

    The eye is an important sense organ for teleost species but can vary greatly depending on the adaption to the habitat, environment during ontogeny and developmental stage of the fish. The eye and retinal morphology of eight commonly caught trawl bycatch species were described: Lepidotrigla mulhalli; Lophonectes gallus; Platycephalus bassensis; Sillago flindersi; Neoplatycephalus richardsoni; Thamnaconus degeni; Parequula melbournensis; and Trachurus declivis. The cone densities ranged from 38 cones per 0.01 mm(2) for S. flindersi to 235 cones per 0.01 mm(2) for P. melbournensis. The rod densities ranged from 22800 cells per 0.01 mm(2) for L. mulhalli to 76634 cells per 0.01 mm(2) for T. declivis and potential visual acuity (based on anatomical measures) ranged from 0.08 in L. gallus to 0.31 in P. melbournensis. Higher rod densities were correlated with maximum habitat depths. Six species had the regular pattern of four double cones arranged around a single cone in the photoreceptor mosaic, while T. declivis had only rows of double cones. P. melbournensis had the greatest potential ability for detecting fine detail based on eye anatomy. The potential visual acuity estimates and rod densities can be applied to suggest the relative detection ability of different species in a commercial fishing context, since vision is a critical sense in an illuminated environment for perceiving an oncoming trawl.

  16. Rpr- and hid-driven cell death in Drosophila photoreceptors.

    Science.gov (United States)

    Hsu, Cheng Da; Adams, Sheila M; O'Tousa, Joseph E

    2002-02-01

    The reaper (rpr) and head involution defective (hid) genes mediate programmed cell death (PCD) during Drosophila development. We show that expression of either rpr or hid under control of a rhodopsin promoter induces rapid cell death of adult photoreceptor cells. Ultrastructural analysis revealed that the dying photoreceptor cells share morphological features with other cells undergoing PCD. The anti-apoptotic baculoviral P35 protein acts downstream of hid activity to suppress the photoreceptor cell death driven by rpr and hid. These results establish that the Drosophila photoreceptors are sensitive to the rpr- and hid-driven cell death pathways.

  17. Rod Photoreceptors Express GPR55 in the Adult Vervet Monkey Retina

    OpenAIRE

    Joseph Bouskila; Pasha Javadi; Christian Casanova; Maurice Ptito; Jean-François Bouchard

    2013-01-01

    Cannabinoids exert their actions mainly through two receptors, the cannabinoid CB1 receptor (CB1R) and cannabinoid CB2 receptor (CB2R). In recent years, the G-protein coupled receptor 55 (GPR55) was suggested as a cannabinoid receptor based on its activation by anandamide and tetrahydrocannabinol. Yet, its formal classification is still a matter of debate. CB1R and CB2R expression patterns are well described for rodent and monkey retinas. In the monkey retina, CB1R has been localized in its n...

  18. Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells

    Directory of Open Access Journals (Sweden)

    Koike Chieko

    2006-03-01

    Full Text Available Abstract Background Sterile alpha motif (SAM domains are ~70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mouse mr-s (major retinal SAM domain protein. Results mr-s is evolutionarily conserved from zebrafish through human, organisms through which the mechanism of photoreceptor development is also highly conserved. Phylogenetic analysis suggests that the SAM domain of mr-s is most closely related to a mouse polyhomeotic (ph ortholog, Mph1/Rae28, which is known as an epigenetic molecule involved in chromatin modifications. These findings provide the possibility that mr-s may play a critical role by regulating gene expression in photoreceptor development. mr-s is preferentially expressed in the photoreceptors at postnatal day 3–6 (P3-6, when photoreceptors undergo terminal differentiation, and in the adult pineal gland. Transcription of mr-s is directly regulated by the cone-rod homeodomain protein Crx. Immunoprecipitation assay showed that the mr-s protein self-associates mainly through the SAM domain-containing region as well as ph. The mr-s protein localizes mainly in the nucleus, when mr-s is overexpressed in HEK293T cells. Moreover, in the luciferase assays, we found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. We revealed that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region. Conclusion We identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and pineal gland. Based on its expression pattern and biochemical analysis

  19. Rhodopsin gene expression determines rod outer segment size and rod cell resistance to a dominant-negative neurodegeneration mutant.

    Directory of Open Access Journals (Sweden)

    Brandee A Price

    Full Text Available Two outstanding unknowns in the biology of photoreceptors are the molecular determinants of cell size, which is remarkably uniform among mammalian species, and the mechanisms of rod cell death associated with inherited neurodegenerative blinding diseases such as retinitis pigmentosa. We have addressed both questions by performing an in vivo titration with rhodopsin gene copies in genetically engineered mice that express only normal rhodopsin or an autosomal dominant allele, encoding rhodopsin with a disease-causing P23H substitution. The results reveal that the volume of the rod outer segment is proportional to rhodopsin gene expression; that P23H-rhodopsin, the most common rhodopsin gene disease allele, causes cell death via a dominant-negative mechanism; and that long term survival of rod cells carrying P23H-rhodopsin can be achieved by increasing the levels of wild type rhodopsin. These results point to promising directions in gene therapy for autosomal dominant neurodegenerative diseases caused by dominant-negative mutations.

  20. Long-term preservation of cone photoreceptors and visual acuity in rd10 mutant mice exposed to continuous environmental enrichment.

    Science.gov (United States)

    Barone, Ilaria; Novelli, Elena; Strettoi, Enrica

    2014-01-01

    In human patients and animal models of retinitis pigmentosa (RP), a gradual loss of rod photoreceptors and decline in scotopic vision are the primary manifestations of the disease. Secondary death of cones and gradual, regressive remodeling of the inner retina follow and progress at different speeds according to the underlying genetic defect. In any case, the final outcome is near-blindness without a conclusive cure yet. We recently reported that environmental enrichment (EE), an experimental manipulation based on exposure to enhanced motor, sensory, and social stimulation, when started at birth, exerts clear beneficial effects on a mouse model of RP, by slowing vision loss. The purpose of this study was to investigate in the same mouse the long-term effects of chronic exposure to an EE and assess the outcome of this manipulation on cone survival, inner retinal preservation, and visual behavior. Two groups of rd10 mutant mice were maintained in an EE or standard (ST) laboratory conditions up to 1 year of age. Then, retinal preservation was assessed with immunocytochemistry, confocal microscopy examination, cone counts, and electron microscopy of the photoreceptor layer, while visual acuity was tested behaviorally with a Prusky water maze. rd10 mice are a model of autosomal recessive RP with a typical rod-cone, center to the periphery pattern of photoreceptor degeneration. They carry a mutation of the rod-specific phosphodiesterase gene and undergo rod death that peaks at around P24, while cone electroretinogram (ERG) is extinct by P60. We previously showed that early exposure to an EE efficiently delays photoreceptor degeneration in these mutants, extending the time window of cone viability and cone-mediated vision well beyond the phase of maximum rod death. Here we find that a maintained EE can delay the degeneration of cones even in the long term. Confocal and electron microscopy examination of the retinas of the rd10 EE and ST mice at 1 year of age showed major

  1. Loss of the metalloprotease ADAM9 leads to cone-rod dystrophy in humans and retinal degeneration in mice.

    Science.gov (United States)

    Parry, David A; Toomes, Carmel; Bida, Lina; Danciger, Michael; Towns, Katherine V; McKibbin, Martin; Jacobson, Samuel G; Logan, Clare V; Ali, Manir; Bond, Jacquelyn; Chance, Rebecca; Swendeman, Steven; Daniele, Lauren L; Springell, Kelly; Adams, Matthew; Johnson, Colin A; Booth, Adam P; Jafri, Hussain; Rashid, Yasmin; Banin, Eyal; Strom, Tim M; Farber, Debora B; Sharon, Dror; Blobel, Carl P; Pugh, Edward N; Pierce, Eric A; Inglehearn, Chris F

    2009-05-01

    Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.

  2. Not just signal shutoff: the protective role of arrestin-1 in rod cells.

    Science.gov (United States)

    Sommer, Martha E; Hofmann, Klaus Peter; Heck, Martin

    2014-01-01

    The retinal rod cell is an exquisitely sensitive single-photon detector that primarily functions in dim light (e.g., moonlight). However, rod cells must routinely survive light intensities more than a billion times greater (e.g., bright daylight). One serious challenge to rod cell survival in daylight is the massive amount of all-trans-retinal that is released by Meta II, the light-activated form of the photoreceptor rhodopsin. All-trans-retinal is toxic, and its condensation products have been implicated in disease. Our recent work has developed the concept that rod arrestin (arrestin-1), which terminates Meta II signaling, has an additional role in protecting rod cells from the consequences of bright light by limiting free all-trans-retinal. In this chapter we will elaborate upon the molecular mechanisms by which arrestin-1 serves as both a single-photon response quencher as well as an instrument of rod cell survival in bright light. This discussion will take place within the framework of three distinct functional modules of vision: signal transduction, the retinoid cycle, and protein translocation.

  3. Control of Ca2+ in rod outer segment disks by light and cyclic GMP.

    Science.gov (United States)

    George, J S; Hagins, W A

    1983-05-26

    Photons absorbed in vertebrate rods and cones probably cause electrochemical changes at the photoreceptor plasma membrane by changing the cytoplasmic concentration of a diffusible transmitter substance, reducing the Na+ current flowing into the outer segment of the cell in the dark, to produce the observed membrane hyperpolarization that is the initial excitatory response. Cyclic GMP has been proposed as the transmitter because a light-activated cyclic GMP phosphodiesterase (PDE) has been found in rod disk membranes and because intracellularly injected cyclic GMP reduces rod membrane potentials. Free Ca2+ has also been proposed because increasing external [Ca2+] quickly and reversibly reduces the dark current and divalent cationophores increase the Ca2+ sensitivity. Ca2+ efflux from rod outer segments (ROS) of intact retinas occurs simultaneously with light responses. Vesicles prepared from ROS disk membranes become more permeable on illumination, releasing trapped ions or molecules, but intact outer segment disks have not previously been found to store sufficient Ca2+ in darkness and to release enough in light to meet the theoretical requirements for control of the dark current by varying cytoplasmic Ca2+ (refs 14-18). We now report experiments that show the required Ca2+ storage and release from rod disk membranes suspended in media containing high-energy phosphate esters and electrolytes approximating the cytoplasmic composition of live rod cells. Cyclic GMP stimulates Ca2+ uptake by ROS disks in such media.

  4. Successful Gene Therapy in the RPGRIP1-deficient Dog: a Large Model of Cone–Rod Dystrophy

    Science.gov (United States)

    Lhériteau, Elsa; Petit, Lolita; Weber, Michel; Le Meur, Guylène; Deschamps, Jack-Yves; Libeau, Lyse; Mendes-Madeira, Alexandra; Guihal, Caroline; François, Achille; Guyon, Richard; Provost, Nathalie; Lemoine, Françoise; Papal, Samantha; El-Amraoui, Aziz; Colle, Marie-Anne; Moullier, Philippe; Rolling, Fabienne

    2014-01-01

    For the development of new therapies, proof-of-concept studies in large animal models that share clinical features with their human counterparts represent a pivotal step. For inherited retinal dystrophies primarily involving photoreceptor cells, the efficacy of gene therapy has been demonstrated in canine models of stationary cone dystrophies and progressive rod–cone dystrophies but not in large models of progressive cone–rod dystrophies, another important cause of blindness. To address the last issue, we evaluated gene therapy in the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)-deficient dog, a model exhibiting a severe cone–rod dystrophy similar to that seen in humans. Subretinal injection of AAV5 (n = 5) or AAV8 (n = 2) encoding the canine Rpgrip1 improved photoreceptor survival in transduced areas of treated retinas. Cone function was significantly and stably rescued in all treated eyes (18–72% of those recorded in normal eyes) up to 24 months postinjection. Rod function was also preserved (22–29% of baseline function) in four of the five treated dogs up to 24 months postinjection. No detectable rod function remained in untreated contralateral eyes. More importantly, treatment preserved bright- and dim-light vision. Efficacy of gene therapy in this large animal model of cone–rod dystrophy provides great promise for human treatment. PMID:24091916

  5. Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol.

    Science.gov (United States)

    Andrews, L D; Cohen, A I

    1983-09-01

    We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin-binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze-fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested

  6. Understanding Cone Photoreceptor Cell Death in Achromatopsia.

    Science.gov (United States)

    Carvalho, Livia S; Vandenberghe, Luk H

    2016-01-01

    Colour vision is only achieved in the presence of healthy and functional cone photoreceptors found in the retina. It is an essential component of human vision and usually the first complaint patients undergoing vision degeneration have is the loss of daylight colour vision. Therefore, an understanding of the biology and basic mechanisms behind cone death under the degenerative state of retinal dystrophies and how the activation of the apoptotic pathway is triggered will provide valuable knowledge. It will also have broader applications for a spectrum of visual disorders and will be critical for future advances in translational research.

  7. Structure and function of the interphotoreceptor matrix surrounding retinal photoreceptor cells.

    Science.gov (United States)

    Ishikawa, Makoto; Sawada, Yu; Yoshitomi, Takeshi

    2015-04-01

    The interphotoreceptor matrix (IPM) is a highly organized structure with interconnected domains surrounding cone and rod photoreceptor cells and extends throughout the subretinal space. Based on known roles of the extracellular matrix in other tissues, the IPM is thought to have several prominent functions including serving as a receptor for growth factors, regulating retinoid transport, participating in cytoskeletal organization in surrounding cells, and regulation of oxygen and nutrient transport. In addition, a number of studies suggest that the IPM also may play a significant role in the etiology of retinal degenerative disorders. In this review, we describe the present knowledge concerning the structure and function of the IPM under physiological and pathological conditions. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Preservation of cone photoreceptors after a rapid yet transient degeneration and remodeling in cone-only Nrl−/− mouse retina

    Science.gov (United States)

    Roger, Jerome E; Ranganath, Keerthi; Zhao, Lian; Cojocaru, Radu I; Brooks, Matthew; Gotoh, Norimoto; Veleri, Shobi; Hiriyanna, Avinash; Rachel, Rivka A; Campos, Maria Mercedes; Fariss, Robert N; Wong, Wai T; Swaroop, Anand

    2012-01-01

    Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor NRL. The loss of Nrl (Nrl−/−) in mice results in a retina with predominantly S-opsin containing cones that exhibit molecular and functional characteristics of WT cones. Here we report that Nrl−/− retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by four months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic ERG. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl−/− mouse illustrates the long-term viability of cones in the absence of rods and RPE defects in a rodless retina. We propose that Nrl−/− retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula. PMID:22238088

  9. Active Brownian rods

    Science.gov (United States)

    Peruani, Fernando

    2016-11-01

    Bacteria, chemically-driven rods, and motility assays are examples of active (i.e. self-propelled) Brownian rods (ABR). The physics of ABR, despite their ubiquity in experimental systems, remains still poorly understood. Here, we review the large-scale properties of collections of ABR moving in a dissipative medium. We address the problem by presenting three different models, of decreasing complexity, which we refer to as model I, II, and III, respectively. Comparing model I, II, and III, we disentangle the role of activity and interactions. In particular, we learn that in two dimensions by ignoring steric or volume exclusion effects, large-scale nematic order seems to be possible, while steric interactions prevent the formation of orientational order at large scales. The macroscopic behavior of ABR results from the interplay between active stresses and local alignment. ABR exhibit, depending on where we locate ourselves in parameter space, a zoology of macroscopic patterns that ranges from polar and nematic bands to dynamic aggregates.

  10. Glutamine inhibits ammonia-induced accumulation of cGMP in rat striatum limiting arginine supply for NO synthesis.

    Science.gov (United States)

    Hilgier, Wojciech; Freśko, Inez; Klemenska, Emilia; Beresewicz, Andrzej; Oja, Simo S; Saransaari, Pirjo; Albrecht, Jan; Zielińska, Magdalena

    2009-07-01

    Brain L-glutamine (Gln) accumulation and increased activity of the NO/cGMP pathway are immediate consequences of acute exposure to ammonia. This study tested whether excess Gln may influence NO and/or cGMP synthesis. Intrastriatal administration of the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine or the system A-specific Gln uptake inhibitor methylaminoisobutyrate increased microdialysate Gln concentration and reduced basal and ammonia-induced NO and cGMP accumulation. Gln applied in vivo (via microdialysis) or in vitro (to rat brain cortical slices) reduced NO and cGMP accumulation in the presence and/or absence of ammonia, but not cGMP synthesis induced by the NO donor sodium nitroprusside. Attenuation of cGMP synthesis by Gln was prevented by administration of L-arginine (Arg). The L-arginine co-substrates of y(+)LAT2 transport system, L-leucine and cyclo-leucine, mimicked the effect of exogenous Gln, suggesting that Gln limits Arg supply for NO synthesis by interfering with y+LAT2-mediated Arg uptake across the cell membrane.

  11. The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling

    KAUST Repository

    Wheeler, Janet I.

    2017-05-08

    The brassinosteroid receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is a member of the leucine rich repeat receptor like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per μg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate BRASSINOSTEROID SIGNALING KINASE 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor. This article is protected by copyright. All rights reserved.

  12. Mutant carbonic anhydrase 4 impairs pH regulation and causes retinal photoreceptor degeneration.

    Science.gov (United States)

    Yang, Zhenglin; Alvarez, Bernardo V; Chakarova, Christina; Jiang, Li; Karan, Goutam; Frederick, Jeanne M; Zhao, Yu; Sauvé, Yves; Li, Xi; Zrenner, Eberhart; Wissinger, Bernd; Hollander, Anneke I Den; Katz, Bradley; Baehr, Wolfgang; Cremers, Frans P; Casey, Joseph R; Bhattacharya, Shomi S; Zhang, Kang

    2005-01-15

    Retina and retinal pigment epithelium (RPE) belong to the metabolically most active tissues in the human body. Efficient removal of acid load from retina and RPE is a critical function mediated by the choriocapillaris. However, the mechanism by which pH homeostasis is maintained is largely unknown. Here, we show that a functional complex of carbonic anhydrase 4 (CA4) and Na+/bicarbonate co-transporter 1 (NBC1) is specifically expressed in the choriocapillaris and that missense mutations in CA4 linked to autosomal dominant rod-cone dystrophy disrupt NBC1-mediated HCO3- transport. Our results identify a novel pathogenic pathway in which a defect in a functional complex involved in maintaining pH balances, but not expressed in retina or RPE, leads to photoreceptor degeneration. The importance of a functional CA4 for survival of photoreceptors implies that CA inhibitors, which are widely used as medications, particularly in the treatment of glaucoma, may have long-term adverse effects on vision.

  13. Control of Rod-Rod Interactions in Poly(3-alkylthiophenes)

    Science.gov (United States)

    Ho, Victor; Boudouris, Bryan W.; Segalman, Rachel A.

    2010-03-01

    Poly(3-hexylthiophene) is a commonly used semiconducting polymer because of its relatively high charge transport ability, low band gap, and solution processiblity. Strong intermolecular interactions lead to the formation of nanofibers during crystallization, which prevents long-range microstructural ordering. We show rod-rod interactions, parameterized by the Maier-Saupe parameter, can be controlled by rational polythiophene side chain design. Effects of side chain passivation are evidenced by a depressed melting temperature and the presence of a liquid crystalline region. Additionally, the Maier-Saupe parameters are estimated for poly(3-dodecylthiophene) and poly(3-ethylhexylthiophene); the relative magnitudes of each are related to the interchain spacings obtained by x-ray diffraction experiments. The systematic tuning of the rod-rod interactions in polythiophenes allows for manipulation of the ratio of Maier-Saupe to the Flory-Huggins parameter, a crucial value in obtaining long-range order in rod-coil block copolymer morphologies.

  14. Luminescence- and nanoparticle-mediated increase of light absorption by photoreceptor cells: Converting UV light to visible light.

    Science.gov (United States)

    Li, Lei; Sahi, Sunil K; Peng, Mingying; Lee, Eric B; Ma, Lun; Wojtowicz, Jennifer L; Malin, John H; Chen, Wei

    2016-02-10

    We developed new optic devices - singly-doped luminescence glasses and nanoparticle-coated lenses that convert UV light to visible light - for improvement of visual system functions. Tb(3+) or Eu(3+) singly-doped borate glasses or CdS-quantum dot (CdS-QD) coated lenses efficiently convert UV light to 542 nm or 613 nm wavelength narrow-band green or red light, or wide-spectrum white light, and thereby provide extra visible light to the eye. In zebrafish (wild-type larvae and adult control animals, retinal degeneration mutants, and light-induced photoreceptor cell degeneration models), the use of Tb(3+) or Eu(3+) doped luminescence glass or CdS-QD coated glass lenses provide additional visible light to the rod and cone photoreceptor cells, and thereby improve the visual system functions. The data provide proof-of-concept for the future development of optic devices for improvement of visual system functions in patients who suffer from photoreceptor cell degeneration or related retinal diseases.

  15. C-opsin expressing photoreceptors in echinoderms.

    Science.gov (United States)

    Ullrich-Lüter, Esther M; D'Aniello, Salvatore; Arnone, Maria I

    2013-07-01

    Today's progress in molecular analysis and, in particular, the increased availability of genome sequences have enabled us to investigate photoreceptor cells (PRCs) in organisms that were formerly inaccessible to experimental manipulation. Our studies of marine non-chordate deuterostomes thus aim to bridge a gap of knowledge regarding the evolution of deuterostome PRCs prior to the emergence of vertebrates' eyes. In this contribution, we will show evidence for expression of a c-opsin photopigment, which, according to our phylogenetic analysis, is closely related to an assemblage of chordate visual c-opsins. An antibody raised against sea urchins' c-opsin protein (Sp-Opsin1) recognizes epitopes in a variety of tissues of different echinoderms. While in sea urchins this c-opsin is expressed in locomotory and buccal tube feet, spines, pedicellaria, and epidermis, in brittlestars and starfish we found the immuno-reaction to be located exclusively in cells within the animals' spines. Structural characteristics of these c-opsin+ PRC types include the close vicinity/connection to nerve strands and a, so far unexplored, conspicuous association with the animals' calcite skeleton, which previously has been hypothesized to play a role in echinoderm photobiology. These features are discussed within the context of the evolution of photoreceptors in echinoderms and in deuterostomes generally.

  16. Funktionelle in vitro-Effekte CAMP/CGMP-modulierender Pharmaka am humanen Detrusormuskel

    Directory of Open Access Journals (Sweden)

    Ückert St

    2002-01-01

    Full Text Available Die zyklischen Nukleotidmonophosphate cAMP und cGMP regulieren als intrazelluläre Second Messenger zahlreiche Gewebe- und Organfunktionen. cAMP und cGMP werden von zellulären Adenylat- und Guanylatzyklasen synthetisiert und von Phosphodiesterasen degradiert, die somit Schlüsselenzyme im Prozeß der Tonusregulation glatter Muskulatur sind. Die Markteinführung des PDE5-Inhibitors Sildenafil (Viagra hat dem Konzept der PDE-Inhibition auch in der Urologie breite Akzeptanz verschafft. Eigene Arbeiten der vergangenen Jahre beschreiben die Präsenz der PDE-Isoenzyme 1, 2, 3, 4 und 5 in der Muskulatur des humanen Detrusors und zeigen das klinische Potential des PDE1-Inhibitors Vinpocetin in der Behandlung der motorischen Dranginkontinenz. Mit dem Ziel der Charakterisierung geeigneter Substanzen für die Pharmakotherapie der Detrusorhyperaktivität haben wir die in vitro-Effekte neuer, selektiver Inhibitoren der PDE des Typs 2, 3 und 5 auf isolierte humane Detrusormuskulatur untersucht und mit denen des Diterpens Forskolin (Aktivator der Adenylatzyklase und der Stickoxid (NO-Donatoren Dihydropyridin (DHP und Na+Nitroprussid (NNP verglichen.

  17. Cuisenaire Rods Go to College.

    Science.gov (United States)

    Chinn, Phyllis; And Others

    1992-01-01

    Presents examples of questions and answers arising from a hands-on and exploratory approach to discrete mathematics using cuisenaire rods. Combinatorial questions about trains formed of cuisenaire rods provide the setting for discovering numerical patterns by experimentation and organizing the results using induction and successive differences.…

  18. Hypergravity differentially modulates cGMP efflux in human melanocytic cells stimulated by nitric oxide and natriuretic peptides

    Science.gov (United States)

    Ivanova, K.; Stieber, C.; Lambers, B.; Block, I.; Krieg, R.; Wellmann, A.; Gerzer, R.

    Nitric oxide NO plays a key role in many patho physiologic processes including inflammation and skin cancer The diverse cellular effects of NO are mainly mediated by activation of the soluble guanylyl cyclase sGC isoform that leads to increases in intracellular cGMP levels whereas the membrane-bound isoforms serve as receptors for natriuretic peptides e g ANP In human skin epidermal melanocytes represent the principal cells for skin pigmentation by synthesizing the pigment melanin Melanin acts as a scavenger for free radicals that may arise during metabolic stress as a result of potentially harmful effects of the environment In previous studies we found that long-term exposure to hypergravity stimulated cGMP efflux in normal human melanocytes NHMs and non-metastatic melanoma cells at least partly by an enhanced expression of the multidrug resistance proteins MRP and cGMP transporters MRP4 5 The present study investigated whether hypergravity generated by centrifugal acceleration may modulate the cGMP efflux in NO-stimulated NHMs and melanoma cells MCs with different metastatic potential The NONOates PAPA-NO and DETA-NO were used as direct NO donors for cell stimulation In the presence of 0 1 mM DETA-NO t 1 2 sim 20 h long-term application of hypergravity up to 5 g for 24 h reduced intracellular cGMP levels by stimulating cGMP efflux in NHMs and non-metastatic MCs in comparison to 1 g whereas exposure to 5 g for 6 h in the presence of 0 1 mM PAPA-NO t 1 2 sim 30 min was not effective The hypergravity-stimulated

  19. CNGB3-achromatopsia clinical trial with CNTF: diminished rod pathway responses with no evidence of improvement in cone function.

    Science.gov (United States)

    Zein, Wadih M; Jeffrey, Brett G; Wiley, Henry E; Turriff, Amy E; Tumminia, Santa J; Tao, Weng; Bush, Ronald A; Marangoni, Dario; Wen, Rong; Wei, Lisa L; Sieving, Paul A

    2014-09-09

    Ciliary neurotrophic factor (CNTF) protects rod photoreceptors from retinal degenerative disease in multiple nonhuman models. Thus far, CNTF has failed to demonstrate rod protection in trials for human retinitis pigmentosa. Recently, CNTF was found to improve cone photoreceptor function in a canine CNGB3 achromatopsia model. This study explores whether this finding translates to humans with CNGB3 achromatopsia. A five-subject, open-label Phase I/II study was initiated by implanting intraocular microcapsules releasing CNTF (nominally 20 ng/d) into one eye each of CNGB3 achromat participants. Fellow eyes served as untreated controls. Subjects were followed for 1 year. Pupil constriction in treated eyes gave evidence of intraocular CNTF release. Additionally, scotopic ERG responses were reduced, and dark-adapted psychophysical absolute thresholds were increased, attributable to diminished rod or rod pathway activity. Optical coherence tomography revealed that the cone-rich fovea underwent structural changes as the foveal hyporeflective zone (HRZ) became diminished in CNTF-treated eyes. No objectively measurable enhancement of cone function was found by assessments of visual acuity, mesopic increment sensitivity threshold, or the photopic ERG. Careful measurements of color hue discrimination showed no change. Nonetheless, subjects reported beneficial changes of visual function in the treated eyes, including reduced light sensitivity and aversion to bright light, which may trace to decreased effective ambient light from the pupillary constriction; further they noted slowed adaptation to darkness, consistent with CNTF action on rod photoreceptors. Ciliary neurotrophic factor did not measurably enhance cone function, which reveals a species difference between human and canine CNGB3 cones in response to CNTF. (ClinicalTrials.gov number, NCT01648452.). Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  20. Abrupt onset of mutations in a developmentally regulated gene during terminal differentiation of post-mitotic photoreceptor neurons in mice.

    Directory of Open Access Journals (Sweden)

    Ivette M Sandoval

    Full Text Available For sensitive detection of rare gene repair events in terminally differentiated photoreceptors, we generated a knockin mouse model by replacing one mouse rhodopsin allele with a form of the human rhodopsin gene that causes a severe, early-onset form of retinitis pigmentosa. The human gene contains a premature stop codon at position 344 (Q344X, cDNA encoding the enhanced green fluorescent protein (EGFP at its 3' end, and a modified 5' untranslated region to reduce translation rate so that the mutant protein does not induce retinal degeneration. Mutations that eliminate the stop codon express a human rhodopsin-EGFP fusion protein (hRho-GFP, which can be readily detected by fluorescence microscopy. Spontaneous mutations were observed at a frequency of about one per retina; in every case, they gave rise to single fluorescent rod cells, indicating that each mutation occurred during or after the last mitotic division. Additionally, the number of fluorescent rods did not increase with age, suggesting that the rhodopsin gene in mature rod cells is less sensitive to mutation than it is in developing rods. Thus, there is a brief developmental window, coinciding with the transcriptional activation of the rhodopsin locus, in which somatic mutations of the rhodopsin gene abruptly begin to appear.

  1. Ontogeny of the photoreceptors in the embryonic retina of the viviparous guppy, Poecilia reticulata P. (Teleostei). An electron-microscopical study.

    Science.gov (United States)

    Kunz, Y W; Ennis, S; Wise, C

    1983-01-01

    Ultrastructural analyses of retinal development in the guppy embryo show that at midgestation all types of photoreceptors are differentiated in the fundus, and at birth differentiation extends over the whole retina. Formation of discs of outer segments is more rapid in rods than in cones. Double cones differentiate simultaneously with long single cones and are formed by the adhesion of two primordial inner segments; short single cones develop last. Wherever cones are differentiated, they are arranged in an adult-type square mosaic. The rods in the embryo, as opposed to the adult, are likewise regularly arranged within the mosaic unit. These results are at variance with the generally held opinion that adult teleosts which possess duplex retinae have larvae with pure cone retinae, and that rods, double cones and mosaics appear in late larval life or only at metamorphosis. In the double cones of the guppy embryo subsurface cisternae develop along the adjoining primordial inner segments. Additionally, regularly distributed subsurface cisternae are formed in the regions of intimate contact of long single cones with double cones and rods. We suggest that the early development of rods and double cones, and a square-mosaic with regular distribution of rods and subsurface cisternae, provide the newly born with a fully functional optical apparatus, especially suited to perception of movements. This is necessary for its survival against predatory, especially maternal, attacks.

  2. Method to Remove Photoreceptors from Wholemount Retina in vitro.

    Science.gov (United States)

    Walston, Steven T; Chang, Yao-Chuan; Weiland, James D; Chow, Robert H

    2017-08-30

    Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct in a wholemount retina preparation because surrounding neurons block the path of the patch pipette. Vertical slice preparations or dissociated retina cell cultures provide access to bipolar cells at the cost of severing lateral connection between neurons. We have developed a technique to remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing access for patch clamp recording. Repeated application and removal of filter paper to the photoreceptor side of an isolated retina effectively and efficiently removes photoreceptor cells and, in degenerate retina, hypertrophied Müller cell endfeet. Live-dead assays applied to neurons remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner retinal neurons in a wholemount retina preparation for investigation of signal processing. A disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The technique may be useful for investigations of extracellular electrical stimulation, photoreceptor DNA analysis, and non-pharmacological removal of light input. Copyright © 2017, Journal of Neurophysiology.

  3. cGMP and nitric oxide modulate thrombin-induced endothelial permeability : Regulation via different pathways in human aortic and umbilical vein endothelial cells

    NARCIS (Netherlands)

    Draijer, R.; Atsma, D.E.; Laarse, A. van der; Hinsbergh, V.W.M. van

    1995-01-01

    Previous studies have demonstrated that cGMP and cAMP reduce the endothelial permeability for fluids and macromolecules when the endothelial permeability is increased by thrombin. In this study, we have investigated the mechanism by which cGMP improves the endothelial barrier function and examined w

  4. Eulerian formulation of elastic rods

    Science.gov (United States)

    Huynen, Alexandre; Detournay, Emmanuel; Denoël, Vincent

    2016-06-01

    In numerous biological, medical and engineering applications, elastic rods are constrained to deform inside or around tube-like surfaces. To solve efficiently this class of problems, the equations governing the deflection of elastic rods are reformulated within the Eulerian framework of this generic tubular constraint defined as a perfectly stiff normal ringed surface. This reformulation hinges on describing the rod-deformed configuration by means of its relative position with respect to a reference curve, defined as the axis or spine curve of the constraint, and on restating the rod local equilibrium in terms of the curvilinear coordinate parametrizing this curve. Associated with a segmentation strategy, which partitions the global problem into a sequence of rod segments either in continuous contact with the constraint or free of contact (except for their extremities), this re-parametrization not only trivializes the detection of new contacts but also transforms these free boundary problems into classic two-points boundary-value problems and suppresses the isoperimetric constraints resulting from the imposition of the rod position at the extremities of each rod segment.

  5. Status of rod consolidation, 1988

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, W.J.

    1989-01-01

    It is estimated that the spent fuel storage pools at some domestic light-water reactors will run out of space before 2003, the year that the US Department of Energy currently predicts it will have a repository available. Of the methods being studied to alleviate the problem, rod consolidation is one of the leading candidates for achieving more efficient use of existing space in spent fuel storage pools. Rod consolidation involves mechanically removing all the fuel rods from the fuel assembly hardware (i.e., the structural components) and placing the fuel rods in a close-packed array in a canister without space grids. A typical goal of rod consolidation systems is to insert the fuel rods from two fuel assemblies into a canister that has the same exterior dimensions as one standard fuel assembly (i.e., to achieve a consolidation or compaction ratio of 2:1) and to compact the nonfuel-bearing structural components from those two fuel assemblies by a factor of 10 to 20. This report provides an overview of the current status of rod consolidation in the United States and a small amount of information on related activities in other countries. 85 refs., 36 figs., 5 tabs.

  6. Action spectra of zebrafish cone photoreceptors.

    Directory of Open Access Journals (Sweden)

    Duco Endeman

    Full Text Available Zebrafish is becoming an increasingly popular model in the field of visual neuroscience. Although the absorption spectra of its cone photopigments have been described, the cone action spectra were still unknown. In this study we report the action spectra of the four types of zebrafish cone photoreceptors, determined by measuring voltage responses upon light stimulation using whole cell patch clamp recordings. A generic template of photopigment absorption spectra was fit to the resulting action spectra in order to establish the maximum absorption wavelength, the A2-based photopigment contribution and the size of the β-wave of each cone-type. Although in general there is close correspondence between zebrafish cone action- and absorbance spectra, our data suggest that in the case of MWS- and LWS-cones there is appreciable contribution of A2-based photopigments and that the β-wave for these cones is smaller than expected based on the absorption spectra.

  7. The dual rod system of amphibians supports colour discrimination at the absolute visual threshold

    Science.gov (United States)

    Yovanovich, Carola A. M.; Koskela, Sanna M.; Nevala, Noora; Kondrashev, Sergei L.

    2017-01-01

    The presence of two spectrally different kinds of rod photoreceptors in amphibians has been hypothesized to enable purely rod-based colour vision at very low light levels. The hypothesis has never been properly tested, so we performed three behavioural experiments at different light intensities with toads (Bufo) and frogs (Rana) to determine the thresholds for colour discrimination. The thresholds of toads were different in mate choice and prey-catching tasks, suggesting that the differential sensitivities of different spectral cone types as well as task-specific factors set limits for the use of colour in these behavioural contexts. In neither task was there any indication of rod-based colour discrimination. By contrast, frogs performing phototactic jumping were able to distinguish blue from green light down to the absolute visual threshold, where vision relies only on rod signals. The remarkable sensitivity of this mechanism comparing signals from the two spectrally different rod types approaches theoretical limits set by photon fluctuations and intrinsic noise. Together, the results indicate that different pathways are involved in processing colour cues depending on the ecological relevance of this information for each task. This article is part of the themed issue ‘Vision in dim light’. PMID:28193811

  8. Automatic cone photoreceptor segmentation using graph theory and dynamic programming.

    Science.gov (United States)

    Chiu, Stephanie J; Lokhnygina, Yuliya; Dubis, Adam M; Dubra, Alfredo; Carroll, Joseph; Izatt, Joseph A; Farsiu, Sina

    2013-06-01

    Geometrical analysis of the photoreceptor mosaic can reveal subclinical ocular pathologies. In this paper, we describe a fully automatic algorithm to identify and segment photoreceptors in adaptive optics ophthalmoscope images of the photoreceptor mosaic. This method is an extension of our previously described closed contour segmentation framework based on graph theory and dynamic programming (GTDP). We validated the performance of the proposed algorithm by comparing it to the state-of-the-art technique on a large data set consisting of over 200,000 cones and posted the results online. We found that the GTDP method achieved a higher detection rate, decreasing the cone miss rate by over a factor of five.

  9. Coupling ex vivo electroporation of mouse retinas and luciferase reporter assays to assess rod-specific promoter activity.

    Science.gov (United States)

    Boulling, Arnaud; Escher, Pascal

    2016-07-01

    Ex vivo electroporation of mouse retinas is an established tool to modulate gene expression and to study cell type-specific gene expression. Here we coupled ex vivo electroporation to luciferase reporter assays to facilitate the study of rod-photoreceptor-specific gene promoters. The activity of the rod-specific proximal bovine rhodopsin promoter was significantly increased in C57BL/6J wild-type retinas at postnatal days 1 and 7 by 3.4-fold and 8.7-fold respectively. In C57BL/6J Nr2e3(rd7/rd7) retinas, where the rod photoreceptor-specific nuclear receptor Nr2e3 is not expressed, a significant increase by 2.5-fold was only observed at postnatal day 7. Cone-specific S-opsin promoter activity was not modulated in C57BL/6J wild-type and Nr2e3(rd7/rd7) retinas. Taken together, we describe an easily implementable protocol to assess rod-specific promoter activity in a physiological context resembling that of the developing postnatal mouse retina.

  10. [Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism].

    Science.gov (United States)

    Makuch, Edyta; Matuszyk, Janusz

    2012-07-20

    PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme's intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.

  11. Relationship between Adaptation of the Folic Acid and the cAMP Mediated cGMP Response in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1983-01-01

    Chemotactic stimulation of post-vegetative Dictyostelium cells with folic acid or aggregative cells with cAMP results in a fast transient cGMP response which peaks at 10 s; basal levels are recovered in about 30-40 s. Stimulation with folic acid or cAMP rapidly desensitizes the cells for equal or lo

  12. Conformation Changes N-terminal Involvement and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

    Energy Technology Data Exchange (ETDEWEB)

    H Wang; H Robinson; H Ke

    2011-12-31

    The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.

  13. Topological mixing with ghost rods

    Science.gov (United States)

    Gouillart, Emmanuelle; Thiffeault, Jean-Luc; Finn, Matthew D.

    2006-03-01

    Topological chaos relies on the periodic motion of obstacles in a two-dimensional flow in order to form nontrivial braids. This motion generates exponential stretching of material lines, and hence efficient mixing. Boyland, Aref, and Stremler [J. Fluid Mech. 403, 277 (2000)] have studied a specific periodic motion of rods that exhibits topological chaos in a viscous fluid. We show that it is possible to extend their work to cases where the motion of the stirring rods is topologically trivial by considering the dynamics of special periodic points that we call “ghost rods”, because they play a similar role to stirring rods. The ghost rods framework provides a new technique for quantifying chaos and gives insight into the mechanisms that produce chaos and mixing. Numerical simulations for Stokes flow support our results.

  14. Aging has the opposite effect on cAMP and cGMP circadian variations in rat Leydig cells.

    Science.gov (United States)

    Baburski, Aleksandar Z; Sokanovic, Srdjan J; Andric, Silvana A; Kostic, Tatjana S

    2017-05-01

    The Leydig cell physiology displays a circadian rhythm driven by a complex interaction of the reproductive axis hormones and circadian system. The final output of this regulatory process is circadian pattern of steroidogenic genes expression and testosterone production. Aging gradually decreases robustness of rhythmic testosterone secretion without change in pattern of LH secretion. Here, we analyzed effect of aging on circadian variation of cAMP and cGMP signaling in Leydig cells. Results showed opposite effect of aging on cAMP and cGMP daily variation. Reduced amplitude of cAMP circadian oscillation was probably associated with changed expression of genes involved in cAMP production (increased circadian pattern of Adcy7, Adcy9, Adcy10 and decreased Adcy3); cAMP degradation (increased Pde4a, decreased Pde8b, canceled rhythm of Pde4d, completely reversed circadian pattern of Pde7b and Pde8a); and circadian expression of protein kinase A subunits (Prkac/PRKAC and Prkar2a). Aging stimulates expression of genes responsible for cGMP production (Nos2, Gucy1a3 and Gucy1b3/GUCYB3) and degradation (Pde5a, Pde6a and Pde6h) but the overall net effect is elevation of cGMP circadian oscillations in Leydig cells. In addition, the expression of cGMP-dependent kinase, Prkg1/PRKG1 is up-regulated. It seems that aging potentiate cGMP- and reduce cAMP-signaling in Leydig cells. Since both signaling pathways affect testosterone production and clockwork in the cells, further insights into these signaling pathways will help to unravel disorders linked to the circadian timing system, aging and reproduction.

  15. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-12-01

    The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

  16. Angiotensin-(1-7) Downregulates Diabetes-Induced cGMP Phosphodiesterase Activation in Rat Corpus Cavernosum

    Science.gov (United States)

    Benter, Ibrahim F.

    2017-01-01

    Molecular mechanisms of the beneficial effects of angiotensin-(1-7), Ang-(1-7), in diabetes-related complications, including erectile dysfunction, remain unclear. We examined the effect of diabetes and/or Ang-(1-7) treatment on vascular reactivity and cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) in corpus cavernosum. Male Wistar rats were grouped as (1) control, (2) diabetic (streptozotocin, STZ, treated), (3) control + Ang-(1-7), and (4) diabetic + Ang-(1-7). Following 3 weeks of Ang-(1-7) treatment subsequent to induction of diabetes, rats were sacrificed. Penile cavernosal tissue was isolated to measure vascular reactivity, PDE gene expression and activity, and levels of p38MAP kinase, nitrites, and cGMP. Carbachol-induced vasorelaxant response after preincubation of corpus cavernosum with PE was significantly attenuated in diabetic rats, and Ang-(1-7) markedly corrected the diabetes-induced impairment. Gene expression and activity of PDE and p38MAP kinase were significantly increased in cavernosal tissue of diabetic rats, and Ang-(1-7) markedly attenuated STZ-induced effects. Ang-(1-7) significantly increased the levels of nitrite and cGMP in cavernosal tissue of control and diabetic rats. Cavernosal tissue of diabetic rats had significantly reduced cGMP levels and Ang-(1-7) markedly prevented the STZ-induced cGMP depletion. This study demonstrates that attenuation of diabetes-induced PDE activity might be one of the key mechanisms in the beneficial effects of Ang-(1-7).

  17. Adaptive Optics Reveals Photoreceptor Abnormalities in Diabetic Macular Ischemia

    Science.gov (United States)

    Nesper, Peter L.; Scarinci, Fabio

    2017-01-01

    Diabetic macular ischemia (DMI) is a phenotype of diabetic retinopathy (DR) associated with chronic hypoxia of retinal tissue. The goal of this prospective observational study was to report evidence of photoreceptor abnormalities using adaptive optics scanning laser ophthalmoscopy (AOSLO) in eyes with DR in the setting of deep capillary plexus (DCP) non-perfusion. Eleven eyes from 11 patients (6 women, age 31–68), diagnosed with DR without macular edema, underwent optical coherence tomography angiography (OCTA) and AOSLO imaging. One patient without OCTA imaging underwent fluorescein angiography to characterize the enlargement of the foveal avascular zone. The parameters studied included photoreceptor heterogeneity packing index (HPi) on AOSLO, as well as DCP non-perfusion and vessel density on OCTA. Using AOSLO, OCTA and spectral domain (SD)-OCT, we observed that photoreceptor abnormalities on AOSLO and SD-OCT were found in eyes with non-perfusion of the DCP on OCTA. All eight eyes with DCP non-flow on OCTA showed photoreceptor abnormalities on AOSLO. Six of the eight eyes also had outer retinal abnormalities on SD-OCT. Three eyes with DR and robust capillary perfusion of the DCP had normal photoreceptors on SD-OCT and AOSLO. Compared to eyes with DR without DCP non-flow, the eight eyes with DCP non-flow had significantly lower HPi (P = 0.013) and parafoveal DCP vessel density (P = 0.016). We found a significant correlation between cone HPi and parafoveal DCP vessel density (r = 0.681, P = 0.030). Using a novel approach with AOSLO and OCTA, this study shows an association between capillary non-perfusion of the DCP and abnormalities in the photoreceptor layer in eyes with DR. This observation is important in confirming the significant contribution of the DCP to oxygen requirements of photoreceptors in DMI, while highlighting the ability of AOSLO to detect subtle photoreceptor changes not always visible on SD-OCT. PMID:28068435

  18. Rhodopsin Forms Nanodomains in Rod Outer Segment Disc Membranes of the Cold-Blooded Xenopus laevis.

    Directory of Open Access Journals (Sweden)

    Tatini Rakshit

    Full Text Available Rhodopsin forms nanoscale domains (i.e., nanodomains in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.

  19. Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila.

    Science.gov (United States)

    Yamaguchi, Satoko; Desplan, Claude; Heisenberg, Martin

    2010-03-23

    The visual systems of most species contain photoreceptors with distinct spectral sensitivities that allow animals to distinguish lights by their spectral composition. In Drosophila, photoreceptors R1-R6 have the same spectral sensitivity throughout the eye and are responsible for motion detection. In contrast, photoreceptors R7 and R8 exhibit heterogeneity and are important for color vision. We investigated how photoreceptor types contribute to the attractiveness of light by blocking the function of certain subsets and by measuring differential phototaxis between spectrally different lights. In a "UV vs. blue" choice, flies with only R1-R6, as well as flies with only R7/R8 photoreceptors, preferred blue, suggesting a nonadditive interaction between the two major subsystems. Flies defective for UV-sensitive R7 function preferred blue, whereas flies defective for either type of R8 (blue- or green-sensitive) preferred UV. In a "blue vs. green" choice, flies defective for R8 (blue) preferred green, whereas those defective for R8 (green) preferred blue. Involvement of all photoreceptors [R1-R6, R7, R8 (blue), R8 (green)] distinguishes phototaxis from motion detection that is mediated exclusively by R1-R6.

  20. Visual ecology and potassium conductances of insect photoreceptors.

    Science.gov (United States)

    Frolov, Roman; Immonen, Esa-Ville; Weckström, Matti

    2016-04-01

    Voltage-activated potassium channels (Kv channels) in the microvillar photoreceptors of arthropods are responsible for repolarization and regulation of photoreceptor signaling bandwidth. On the basis of analyzing Kv channels in dipteran flies, it was suggested that diurnal, rapidly flying insects predominantly express sustained K(+) conductances, whereas crepuscular and nocturnally active animals exhibit strongly inactivating Kv conductances. The latter was suggested to function for minimizing cellular energy consumption. In this study we further explore the evolutionary adaptations of the photoreceptor channelome to visual ecology and behavior by comparing K(+) conductances in 15 phylogenetically diverse insects, using patch-clamp recordings from dissociated ommatidia. We show that rapid diurnal flyers such as the blowfly (Calliphora vicina) and the honeybee (Apis mellifera) express relatively large noninactivating Kv conductances, conforming to the earlier hypothesis in Diptera. Nocturnal and/or slow-moving species do not in general exhibit stronger Kv conductance inactivation in the physiological membrane voltage range, but the photoreceptors in species that are known to rely more on vision behaviorally had higher densities of sustained Kv conductances than photoreceptors of less visually guided species. No statistically significant trends related to visual performance could be identified for the rapidly inactivating Kv conductances. Counterintuitively, strong negative correlations were observed between photoreceptor capacitance and specific membrane conductance for both sustained and inactivating fractions of Kv conductance, suggesting insignificant evolutionary pressure to offset negative effects of high capacitance on membrane filtering with increased conductance.

  1. Rod and cone pathway signalling is altered in the P2X7 receptor knock out mouse.

    Directory of Open Access Journals (Sweden)

    Kirstan A Vessey

    Full Text Available The P2X7 receptor (P2X7-R is expressed in the retina and brain and has been implicated in neurodegenerative diseases. However, whether it is expressed by neurons and plays a role as a neurotransmitter receptor has been the subject of controversy. In this study, we first show that the novel vesicular transporter for ATP, VNUT, is expressed in the retina, verifying the presence of the molecular machinery for ATP to act as neurotransmitter at P2X7-Rs. Secondly we show the presence of P2X7-R mRNA and protein in the retina and cortex and absence of the full length variant 1 of the receptor in the P2X7-R knock out (P2X7-KO mouse. The role of the P2X7-R in neuronal function of the retina was assessed by comparing the electroretinogram response of P2X7-KO with WT mice. The rod photoreceptor response was found to be similar, while both rod and cone pathway post-photoreceptor responses were significantly larger in P2X7-KO mice. This suggests that activation of P2X7-Rs modulates output of second order retinal neurons. In line with this finding, P2X7-Rs were found in the outer plexiform layer and on inner retinal cell classes, including horizontal, amacrine and ganglion cells. The receptor co-localized with conventional synapses in the IPL and was expressed on amacrine cells post-synaptic to rod bipolar ribbon synapses. In view of the changes in visual function in the P2X7-KO mouse and the immunocytochemical location of the receptor in the normal retina, it is likely the P2X7-R provides excitatory input to photoreceptor terminals or to inhibitory cells that shape both the rod and cone pathway response.

  2. Ablation of EYS in zebrafish causes mislocalisation of outer segment proteins, F-actin disruption and cone-rod dystrophy

    Science.gov (United States)

    Lu, Zhaojing; Hu, Xuebin; Liu, Fei; Soares, Dinesh C.; Liu, Xiliang; Yu, Shanshan; Gao, Meng; Han, Shanshan; Qin, Yayun; Li, Chang; Jiang, Tao; Luo, Daji; Guo, An-Yuan; Tang, Zhaohui; Liu, Mugen

    2017-01-01

    Mutations in EYS are associated with autosomal recessive retinitis pigmentosa (arRP) and autosomal recessive cone-rod dystrophy (arCRD) however, the function of EYS and the molecular mechanisms of how these mutations cause retinal degeneration are still unclear. Because EYS is absent in mouse and rat, and the structure of the retina differs substantially between humans and Drosophila, we utilised zebrafish as a model organism to study the function of EYS in the retina. We constructed an EYS-knockout zebrafish-line by TALEN technology which showed visual impairment at an early age, while the histological and immunofluorescence assays indicated the presence of progressive retinal degeneration with a cone predominately affected pattern. These phenotypes recapitulate the clinical manifestations of arCRD patients. Furthermore, the EYS−/− zebrafish also showed mislocalisation of certain outer segment proteins (rhodopsin, opn1lw, opn1sw1, GNB3 and PRPH2), and disruption of actin filaments in photoreceptors. Protein mislocalisation may, therefore, disrupt the function of cones and rods in these zebrafish and cause photoreceptor death. Collectively, these results point to a novel role for EYS in maintaining the morphological structure of F-actin and in protein transport, loss of this function might be the trigger for the resultant cellular events that ultimately lead to photoreceptor death. PMID:28378834

  3.  Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism

    OpenAIRE

    Edyta Makuch; Janusz Matuszyk

    2012-01-01

     PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regio...

  4. The Third ATLAS ROD Workshop

    CERN Multimedia

    Poggioli, L.

    A new-style Workshop After two successful ATLAS ROD Workshops dedicated to the ROD hardware and held at the Geneva University in 1998 and in 2000, a new style Workshop took place at LAPP in Annecy on November 14-15, 2002. This time the Workshop was fully dedicated to the ROD-TDAQ integration and software in view of the near future integration activities of the final RODs for the detector assembly and commissioning. More precisely, the aim of this workshop was to get from the sub-detectors the parameters needed for T-DAQ, as well as status and plans from ROD builders. On the other hand, what was decided and assumed had to be stated (like EB decisions and URDs), and also support plans. The Workshop gathered about 70 participants from all ATLAS sub-detectors and the T-DAQ community. The quite dense agenda allowed nevertheless for many lively discussions, and for a dinner in the old town of Annecy. The Sessions The Workshop was organized in five main sessions: Assumptions and recommendations Sub-de...

  5. Cone photoreceptors are the main targets for gene therapy of NPHP5 (IQCB1) or NPHP6 (CEP290) blindness: generation of an all-cone Nphp6 hypomorph mouse that mimics the human retinal ciliopathy.

    Science.gov (United States)

    Cideciyan, Artur V; Rachel, Rivka A; Aleman, Tomas S; Swider, Malgorzata; Schwartz, Sharon B; Sumaroka, Alexander; Roman, Alejandro J; Stone, Edwin M; Jacobson, Samuel G; Swaroop, Anand

    2011-04-01

    Leber congenital amaurosis (LCA), a severe autosomal recessive childhood blindness, is caused by mutations in at least 15 genes. The most common molecular form is a ciliopathy due to NPHP6 (CEP290) mutations and subjects have profound loss of vision. A similarly severe phenotype occurs in the related ciliopathy NPHP5 (IQCB1)-LCA. Recent success of retinal gene therapy in one form of LCA prompted the question whether we know enough about human NPHP5 and NPHP6 disease to plan such treatment. We determined that there was early-onset rapid degeneration of rod photoreceptors in young subjects with these ciliopathies. Rod outer segment (OS) lamination, when detectable, was disorganized. Retinal pigment epithelium lipofuscin accumulation indicated that rods had existed in the past in most subjects. In contrast to early rod losses, the all-cone human fovea in NPHP5- and NPHP6-LCA of all ages retained cone nuclei, albeit with abnormal inner segments and OS. The rd16 mouse, carrying a hypomorphic Nphp6 allele, was a good model of the rod-dominant human extra-foveal retina. Rd16 mice showed normal genesis of photoreceptors, including the formation of cilia, followed by abnormal elaboration of OS and rapid degeneration. To produce a model of the all-cone human fovea in NPHP6-LCA, we generated rd16;Nrl-/- double-mutant mice. They showed substantially retained cone photoreceptors with disproportionate cone function loss, such as in the human disease. NPHP5- and NPHP6-LCA across a wide age spectrum are thus excellent candidates for cone-directed gene augmentation therapy, and the rd16;Nrl-/- mouse is an appropriate model for pre-clinical proof-of-concept studies.

  6. Human neural progenitor cells promote photoreceptor survival in retinal explants.

    Science.gov (United States)

    Englund-Johansson, Ulrica; Mohlin, Camilla; Liljekvist-Soltic, Ingela; Ekström, Per; Johansson, Kjell

    2010-02-01

    Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of

  7. Binding of Ca2+ to Glutamic Acid-Rich Polypeptides from the Rod Outer Segment

    Science.gov (United States)

    Haber-Pohlmeier, S.; Abarca-Heidemann, K.; Körschen, H. G.; Dhiman, H. Kaur; Heberle, J.; Schwalbe, H.; Klein-Seetharaman, J.; Kaupp, U. B.; Pohlmeier, A.

    2007-01-01

    Rod photoreceptors contain three different glutamic acid-rich proteins (GARPs) that have been proposed to control the propagation of Ca2+ from the site of its entry at the cyclic nucleotide-gated channel to the cytosol of the outer segment. We tested this hypothesis by measuring the binding of Ca2+ to the following five constructs related to GARPs of rod photoreceptors: a 32-mer peptide containing 22 carboxylate groups, polyglutamic acid, a recombinant segment comprising 73 carboxylate groups (GLU), GARP1, and GARP2. Ca2+ binding was investigated by means of a Ca2+-sensitive electrode. In all cases, Ca2+ binds with low affinity; the half-maximum binding constant K1/2 ranges from 6 to 16 mM. The binding stoichiometry between Ca2+ ions and carboxylic groups is ∼1:1; an exception is GARP2, where a binding stoichiometry of ∼1:2 was found. Hydrodynamic radii of 1.6, 2.8, 3.3, 5.7, and 6.7 nm were determined by dynamic light scattering for the 32-mer, polyglutamic acid, GLU, GARP2, and GARP1 constructs, respectively. These results suggest that the peptides as well as GARP1 and GARP2 do not adopt compact globular structures. We conclude that the structures should be regarded as loose coils with low-affinity, high-capacity Ca2+ binding. PMID:17218469

  8. Large-conductance calcium-activated potassium channels facilitate transmitter release in salamander rod synapse.

    Science.gov (United States)

    Xu, Jian Wei; Slaughter, Malcolm M

    2005-08-17

    Large-conductance calcium-activated potassium (BK) channels are colocalized with calcium channels at sites of exocytosis at the presynaptic terminals throughout the nervous system. It is expected that their activation would provide negative feedback to transmitter release, but the opposite is sometimes observed. Attempts to resolve this apparent paradox based on alterations in action potential waveform have been ambiguous. In an alternative approach, we investigated the influence of this channel on neurotransmitter release in a nonspiking neuron, the salamander rod photoreceptors. Surprisingly, the BK channel facilitates calcium-mediated transmitter release from rods. The two presynaptic channels form a positive coupled loop. Calcium influx activates the BK channel current, leading to potassium efflux that increases the calcium current. The normal physiological voltage range of the rod is well matched to the dynamics of this positive loop. When the rod is further depolarized, then the hyperpolarizing BK channel current exceeds its facilitatory effect, causing truncation of transmitter release. Thus, the calcium channel-BK channel linkage performs two functions at the synapse: nonlinear potentiator and safety brake.

  9. Topological Optimization of Rod Mixers

    Science.gov (United States)

    Finn, Matthew D.; Thiffeault, Jean-Luc

    2006-11-01

    Stirring of fluid with moving rods is necessary in many practical applications to achieve homogeneity. These rods are topological obstacles that force stretching of fluid elements. The resulting stretching and folding is commonly observed as filaments and striations, and is a precursor to mixing. In a space-time diagram, the trajectories of the rods form a braid [1], and the properties of this braid impose a minimal complexity in the flow. We discuss how optimal mixing protocols can be obtained by a judicious choice of braid, and how these protocols can be implemented using simple gearing [2].[12pt] [1] P. L. Boyland, H. Aref, and M. A. Stremler, JFM 403, 277 (2000).[8pt] [2] J.-L. Thiffeault and M. D. Finn, http://arxiv.org/nlin/0603003

  10. Advanced gray rod control assembly

    Energy Technology Data Exchange (ETDEWEB)

    Drudy, Keith J; Carlson, William R; Conner, Michael E; Goldenfield, Mark; Hone, Michael J; Long, Jr., Carroll J; Parkinson, Jerod; Pomirleanu, Radu O

    2013-09-17

    An advanced gray rod control assembly (GRCA) for a nuclear reactor. The GRCA provides controlled insertion of gray rod assemblies into the reactor, thereby controlling the rate of power produced by the reactor and providing reactivity control at full power. Each gray rod assembly includes an elongated tubular member, a primary neutron-absorber disposed within the tubular member said neutron-absorber comprising an absorber material, preferably tungsten, having a 2200 m/s neutron absorption microscopic capture cross-section of from 10 to 30 barns. An internal support tube can be positioned between the primary absorber and the tubular member as a secondary absorber to enhance neutron absorption, absorber depletion, assembly weight, and assembly heat transfer characteristics.

  11. Bestrophinopathy: An RPE-photoreceptor interface disease.

    Science.gov (United States)

    Guziewicz, Karina E; Sinha, Divya; Gómez, Néstor M; Zorych, Kathryn; Dutrow, Emily V; Dhingra, Anuradha; Mullins, Robert F; Stone, Edwin M; Gamm, David M; Boesze-Battaglia, Kathleen; Aguirre, Gustavo D

    2017-01-19

    Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies.

  12. Potentiation of cGMP signaling increases oxygen delivery and oxidative metabolism in contracting skeletal muscle of older but not young humans

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Piil, Peter Bergmann; Egelund, Jon;

    2015-01-01

    regulation remain unresolved. Cyclic guanosine monophosphate (cGMP) is one of the main second messengers that mediate smooth muscle vasodilation and alterations in cGMP signaling could, therefore, be one mechanism by which skeletal muscle perfusion is impaired with advancing age. The current study aimed...... to evaluate the effect of inhibiting the main enzyme involved in cGMP degradation, phosphodiesterase 5 (PDE5), on blood flow and O2 delivery in contracting skeletal muscle of young and older humans. A group of young (23 ± 1 years) and a group of older (72 ± 2 years) male human subjects performed submaximal...... in the older subjects correlated with the increase in leg O2 uptake (r (2) = 0.843). These findings suggest an insufficient O2 delivery to the contracting skeletal muscle of aged individuals and that reduced cGMP availability is a novel mechanism underlying impaired skeletal muscle perfusion with advancing age....

  13. Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics.

    Science.gov (United States)

    Cideciyan, Artur V; Aleman, Tomas S; Boye, Sanford L; Schwartz, Sharon B; Kaushal, Shalesh; Roman, Alejandro J; Pang, Ji-Jing; Sumaroka, Alexander; Windsor, Elizabeth A M; Wilson, James M; Flotte, Terence R; Fishman, Gerald A; Heon, Elise; Stone, Edwin M; Byrne, Barry J; Jacobson, Samuel G; Hauswirth, William W

    2008-09-30

    The RPE65 gene encodes the isomerase of the retinoid cycle, the enzymatic pathway that underlies mammalian vision. Mutations in RPE65 disrupt the retinoid cycle and cause a congenital human blindness known as Leber congenital amaurosis (LCA). We used adeno-associated virus-2-based RPE65 gene replacement therapy to treat three young adults with RPE65-LCA and measured their vision before and up to 90 days after the intervention. All three patients showed a statistically significant increase in visual sensitivity at 30 days after treatment localized to retinal areas that had received the vector. There were no changes in the effect between 30 and 90 days. Both cone- and rod-photoreceptor-based vision could be demonstrated in treated areas. For cones, there were increases of up to 1.7 log units (i.e., 50 fold); and for rods, there were gains of up to 4.8 log units (i.e., 63,000 fold). To assess what fraction of full vision potential was restored by gene therapy, we related the degree of light sensitivity to the level of remaining photoreceptors within the treatment area. We found that the intervention could overcome nearly all of the loss of light sensitivity resulting from the biochemical blockade. However, this reconstituted retinoid cycle was not completely normal. Resensitization kinetics of the newly treated rods were remarkably slow and required 8 h or more for the attainment of full sensitivity, compared with gene therapy.

  14. Effect of aerobic exercise training on cGMP levels and blood pressure in treated hypertensive postmenopausal women

    Directory of Open Access Journals (Sweden)

    Iane P Novais

    Full Text Available Abstract The second messenger cGMP has been largely studied as a therapeutic target in a variety of disorders such as erectile dysfunction, arterial hypertension and heart failure. Evidence has shown thatcGMP activators are less efficient in estrogen-deficiency animals, but no studies exist involving non-pharmacological approacheson NO/cGMP signaling pathway in hypertensive postmenopausal women. The aim of this study is to examine NO/cGMP pathway, redox state and blood pressure in trained treatedhypertensive (HT postmenopausal women comparing with normotensive (NT group. The rationale for that is most of HT patients is encouraged by physician to perform exercise associated with pharmacological treatments.Aerobic exercise training (AET consisted of 24 sessions, 3 times/week.Parameters were evaluated at baseline and after AET for both groups (HT=28; NT=33.In treatedHT group, AET was significantly effective in increasing cGMP concentrations (28% accompanied by an up-regulation of SOD (97% and catalase activity (37%. In NT group, we found an increasein SOD activity (58%. TreatedHT postmenopausal women were still responsive to AET increasing cGMP levels and up-regulating antioxidant system. It should also be emphasized that these findings provide information on the circulating biomarkers that might delay the developing of cardiovascular events in this particular population.

  15. The Nitric oxide/CGMP/KATP pathway mediates systemic and central antinociception induced by resistance exercise in rats.

    Science.gov (United States)

    Galdino, Giovane S; Xavier, Carlos H; Almeida, Renato; Silva, Grazielle; Fontes, Marcos A; Menezes, Gustavo; Duarte, Igor D; Perez, Andrea C

    2015-01-01

    Resistance exercise (RE) is characterized to increase strength, tone, mass, and/or muscular endurance and also for produces many beneficial effects, such as blood pressure and osteoporosis reduction, diabetes mellitus control, and analgesia. However, few studies have investigated endogenous mechanisms involved in the RE-induced analgesia. Thus, the aim of this study was evaluate the role of the NO/CGMP/KATP pathway in the antinociception induced by RE. Wistar rats were submitted to acute RE in a weight-lifting model. The nociceptive threshold was measured by mechanical nociceptive test (paw-withdrawal). To investigate the involvement of the NO/CGMP/KATP pathway the following nitric oxide synthase (NOS) non-specific and specific inhibitors were used: N-nitro-l-arginine (NOArg), Aminoguanidine, N5-(1-Iminoethyl)-l-ornithine dihydrocloride (l-NIO), Nω-Propyl-l-arginine (l-NPA); guanylyl cyclase inhibitor, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ); and KATP channel blocker, Glybenclamide; all administered subcutaneously, intrathecally and intracerebroventricularly. Plasma and cerebrospinal fluid (CSF) nitrite levels were determined by spectrophotometry. The RE protocol produced antinociception, which was significantly reversed by NOS specific and unspecific inhibitors, guanylyl cyclase inhibitor (ODQ) and KATP channel blocker (Glybenclamide). RE was also responsible for increasing nitrite levels in both plasma and CSF. These finding suggest that the NO/CGMP/KATP pathway participates in antinociception induced by RE.

  16. Effects of Na/sup +/ on ultraviolet light-induced photorelaxation and c-GMP levels in rabbit aorta

    Energy Technology Data Exchange (ETDEWEB)

    Aceto, J.F.; Raffa, R.B.; Tallarida, R.J.

    1986-03-05

    Isolated strips of rabbit aorta in a state of drug-induced contraction relax reversibly when irradiated with ultraviolet light. The authors previously found that the magnitude of the photorelaxation progressively diminished as the extracellular Na/sup +/ ion concentration was reduced from 145 mM to 85 mM. At 85 mM Na/sup +/, there was minimal photorelaxation, even though the preparation continued to respond to vasoconstricting agents. The reduction in photosensitivity is not an osmotic effect because restoration of osmolarity did not restore photosensitivity. Neither the mechanism underlaying photorelaxation nor its modification by Na/sup +/ is precisely known. In order to examine these further the authors measured cyclic GMP levels in the absence and presence of UV light at both normal and reduced Na/sup +/ levels. At 145 mM Na/sup +/, irradiation resulted in an increase of cGMP from 0.299 to 0.717 fmole/..mu..g protein. At 85 mM Na/sup +/, the corresponding levels were 0.541 and 1.24 fmole/..mu..g protein. Thus, cGMP levels increase (approximately double) with UV irradiation at both reduced and normal Na/sup +/ concentrations even though there is little or no photorelaxation in the reduced Na/sup +/ environment. The reduction in Na/sup +/ may uncouple a link between cGMP elevation and cytoplasmic calcium in the aortic cell.

  17. Co-crystal structures of PKG Iβ (92-227 with cGMP and cAMP reveal the molecular details of cyclic-nucleotide binding.

    Directory of Open Access Journals (Sweden)

    Jeong Joo Kim

    Full Text Available Cyclic GMP-dependent protein kinases (PKGs are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively.We have investigated the mechanism of cyclic nucleotide binding to PKG by determining crystal structures of the amino-terminal cyclic nucleotide-binding domain (CNBD-A of human PKG I bound to either cGMP or cAMP. We also determined the structure of CNBD-A in the absence of bound nucleotide. The crystal structures of CNBD-A with bound cAMP or cGMP reveal that cAMP binds in either syn or anti configurations whereas cGMP binds only in a syn configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP.Our findings suggest that CNBD-A binds cGMP in the syn conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity.

  18. Angiotensin II increases phosphodiesterase 5A expression in vascular smooth muscle cells: A mechanism by which angiotensin II antagonizes cGMP signaling

    Science.gov (United States)

    Kim, Dongsoo; Aizawa, Toru; Wei, Heng; Pi, Xinchun; Rybalkin, Sergei D.; Berk, Bradford C.; Yan, Chen

    2014-01-01

    Angiotensin II (Ang II) and nitric oxide (NO)/natriuretic peptide (NP) signaling pathways mutually regulate each other. Imbalance of Ang II and NO/NP has been implicated in the pathophysiology of many vascular diseases. cGMP functions as a key mediator in the interaction between Ang II and NO/NP. Cyclic nucleotide phosphodiesterase 5A (PDE5A) is important in modulating cGMP signaling by hydrolyzing cGMP in vascular smooth muscle cells (VSMC). Therefore, we examined whether Ang II negatively modulates intracellular cGMP signaling in VSMC by regulating PDE5A. Ang II rapidly and transiently increased PDE5A mRNA levels in rat aortic VSMC. Upregulation of PDE5A mRNA was associated with a time-dependent increase of both PDE5 protein expression and activity. Increased PDE5A mRNA level was transcription-dependent and mediated by the Ang II type 1 receptor. Ang II-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was essential for Ang II-induced PDE5A upregulation. Pretreatment of VSMC with Ang II inhibited C-type NP (CNP) stimulated cGMP signaling, such as cGMP dependent protein kinase (PKG)-mediated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP). Ang II-mediated inhibition of PKG was blocked when PDE5 activity was decreased by selective PDE5 inhibitors, suggesting that upregulation of PDE5A expression is an important mechanism for Ang II to attenuate cGMP signaling. PDE5A may also play a critical role in the growth promoting effects of Ang II because inhibition of PDE5A activity significantly decreased Ang II-stimulated VSMC growth. These observations establish a new mechanism by which Ang II antagonizes cGMP signaling and stimulates VSMC growth. PMID:15623434

  19. The adult retinal stem cell is a rare cell in the ciliary epithelium whose progeny can differentiate into photoreceptors

    Directory of Open Access Journals (Sweden)

    Brian G. Ballios

    2012-02-01

    Self-renewing, multipotential retinal stem cells (RSCs reside in the pigmented ciliary epithelium of the peripheral retina in adult mammals. RSCs can give rise to rhodopsin positive-cells, which can integrate into early postnatal retina, and represent a potentially useful option for cellular therapy. The ability to purify a stem cell population and direct the differentiation toward a particular cell lineage is a challenge facing the application of stem cells in regenerative medicine. Here we use cell sorting to prospectively enrich mouse RSCs based on size, granularity and low expression of P-cadherin and demonstrate that only rare cells with defined properties proliferate to form colonies. We show that clonally-derived mouse and human RSC progeny are multipotent and can differentiate into mature rhodopsin-positive cells with high efficiency using combinations of exogenous culture additives known to influence neural retinal development, including taurine and retinoic acid. This directed RSC differentiation follows the temporal sequence of photoreceptor differentiation in vivo, and the cells exhibit morphology, protein and gene expression consistent with primary cultures of rods in vitro. These results demonstrate that the RSC, an adult stem cell, can be enriched and directed to produce photoreceptors as a first step toward a targeted cell replacement strategy to treat retinal degenerative disease.

  20. The two-step development of a duplex retina involves distinct events of cone and rod neurogenesis and differentiation.

    Science.gov (United States)

    Valen, Ragnhild; Eilertsen, Mariann; Edvardsen, Rolf Brudvik; Furmanek, Tomasz; Rønnestad, Ivar; van der Meeren, Terje; Karlsen, Ørjan; Nilsen, Tom Ole; Helvik, Jon Vidar

    2016-08-15

    Unlike in mammals, persistent postembryonic retinal growth is a characteristic feature of fish, which includes major remodeling events that affect all cell types including photoreceptors. Consequently, visual capabilities change during development, where retinal sensitivity to different wavelengths of light (photopic vision), -and to limited photons (scotopic vision) are central capabilities for survival. Differently from well-established model fish, Atlantic cod has a prolonged larval stage where only cone photoreceptors are present. Rods do not appear until juvenile transition (metamorphosis), a hallmark of indirect developing species. Previously we showed that whole gene families of lws (red-sensitive) and sws1 (UV-sensitive) opsins have been lost in cod, while rh2a (green-sensitive) and sws2 (blue-sensitive) genes have tandem duplicated. Here, we provide a comprehensive characterization of a two-step developing duplex retina in Atlantic cod. The study focuses on cone subtype dynamics and delayed rod neurogenesis and differentiation in all cod life stages. Using transcriptomic and histological approaches we show that different opsins disappear in a topographic manner during development where central to peripheral retina is a key axis of expressional change. Early cone differentiation was initiated in dorso-temporal retina different from previously described in fish. Rods first appeared during initiation of metamorphosis and expression of the nuclear receptor transcription factor nr2e3-1, suggest involvement in rod specification. The indirect developmental strategy thus allows for separate studies of cones and rods development, which in nature correlates with visual changes linked to habitat shifts. The clustering of key retinal genes according to life stage, suggests that Atlantic cod with its sequenced genome may be an important resource for identification of underlying factors required for development and function of photopic and scotopic vision.

  1. Now that you want to take your HIV/AIDS vaccine/biological product research concept into the clinic: what are the "cGMP"?

    Science.gov (United States)

    Sheets, Rebecca L; Rangavajhula, Vijaya; Pullen, Jeffrey K; Butler, Chris; Mehra, Vijay; Shapiro, Stuart; Pensiero, Michael

    2015-04-08

    The Division of AIDS Vaccine Research Program funds the discovery and development of HIV/AIDS vaccine candidates. Basic researchers, having discovered a potential vaccine in the laboratory, next want to take that candidate into the clinic to test the concept in humans, to see if it translates. Many of them have heard of "cGMP" and know that they are supposed to make a "GMP product" to take into the clinic, but often they are not very familiar with what "cGMP" means and why these good practices are so important. As members of the Vaccine Translational Research Branch, we frequently get asked "can't we use the material we made in the lab in the clinic?" or "aren't Phase 1 studies exempt from cGMP?" Over the years, we have had many experiences where researchers or their selected contract manufacturing organizations have not applied an appropriate degree of compliance with cGMP suitable for the clinical phase of development. We share some of these experiences and the lessons learned, along with explaining the importance of cGMP, just what cGMP means, and what they can assure, in an effort to de-mystify this subject and facilitate the rapid and safe translational development of HIV vaccines.

  2. Control rods in LMFBRs: a physics assessment

    Energy Technology Data Exchange (ETDEWEB)

    McFarlane, H.F.; Collins, P.J.

    1982-08-01

    This physics assessment is based on roughly 300 control rod worth measurements in ZPPR from 1972 to 1981. All ZPPR assemblies simulated mixed-oxide LMFBRs, representing sizes of 350, 700, and 900 MWe. Control rod worth measurements included single rods, various combinations of rods, and Ta and Eu rods. Additional measurements studied variations in B/sub 4/C enrichment, rod interaction effects, variations in rod geometry, neutron streaming in sodium-filled channels, and axial worth profiles. Analyses were done with design-equivalent methods, using ENDF/B Version IV data. Some computations for the sensitivities to approximations in the methods have been included. Comparisons of these analyses with the experiments have allowed the status of control rod physics in the US to be clearly defined.

  3. PHOTORECEPTOR DEGENERATION IN A MOUNTAIN LION CUB (PUMA CONCOLOR).

    Science.gov (United States)

    DiSalvo, Andrew R; Reilly, Christopher M; Wiggans, K Tomo; Woods, Leslie W; Wack, Ray F; Clifford, Deana L

    2016-12-01

    An orphaned 4-mo-old female mountain lion cub ( Puma concolor ) was captured along the coastline in Montaña de Oro State Park in Los Osos, California, USA. Following suspicion that the cub was visually impaired, ophthalmic examination revealed diffuse bilateral retinal atrophy. Due to a poor prognosis, humane euthanasia was elected. Necropsy and histopathological findings were consistent with photoreceptor degeneration. Based on the cub's signalment, history, and histopathology, a genetic or nutritional etiology was suspected, with the former etiology more strongly supported. To the authors' knowledge, this is the first report of photoreceptor degeneration in a wild felid and should be considered in cases of blindness.

  4. Solid-state-laser-rod holder

    Science.gov (United States)

    Gettemy, D.J.; Barnes, N.P.; Griggs, J.E.

    1981-08-11

    The disclosure relates to a solid state laser rod holder comprising Invar, copper tubing, and epoxy joints. Materials and coefficients of expansion of the components of the holder combine with the rod to produce a joint which will give before the rod itself will. The rod may be lased at about 70 to 80/sup 0/K and returned from such a temperature to room temperature repeatedly without its or the holder's destruction.

  5. Gene therapy into photoreceptors and Müller glial cells restores retinal structure and function in CRB1 retinitis pigmentosa mouse models.

    Science.gov (United States)

    Pellissier, Lucie P; Quinn, Peter M; Alves, C Henrique; Vos, Rogier M; Klooster, Jan; Flannery, John G; Heimel, J Alexander; Wijnholds, Jan

    2015-06-01

    Mutations in the Crumbs-homologue-1 (CRB1) gene lead to severe recessive inherited retinal dystrophies. Gene transfer therapy is the most promising cure for retinal dystrophies and has primarily been applied for recessive null conditions via a viral gene expression vector transferring a cDNA encoding an enzyme or channel protein, and targeting expression to one cell type. Therapy for the human CRB1 disease will be more complex, as CRB1 is a structural and signaling transmembrane protein present in three cell classes: Müller glia, cone and rod photoreceptors. In this study, we applied CRB1 and CRB2 gene therapy vectors in Crb1-retinitis pigmentosa mouse models at mid-stage disease. We tested if CRB expression restricted to Müller glial cells or photoreceptors or co-expression in both is required to recover retinal function. We show that targeting both Müller glial cells and photoreceptors with CRB2 ameliorated retinal function and structure in Crb1 mouse models. Surprisingly, targeting a single cell type or all cell types with CRB1 reduced retinal function. We show here the first pre-clinical studies for CRB1-related eye disorders using CRB2 vectors and initial elucidation of the cellular mechanisms underlying CRB1 function.

  6. 21 CFR 876.4270 - Colostomy rod.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Colostomy rod. 876.4270 Section 876.4270 Food and... GASTROENTEROLOGY-UROLOGY DEVICES Surgical Devices § 876.4270 Colostomy rod. (a) Identification. A colostomy rod is a device used during the loop colostomy procedure. A loop of colon is surgically brought out...

  7. Solitary waves on nonlinear elastic rods. II

    DEFF Research Database (Denmark)

    Sørensen, Mads Peter; Christiansen, Peter Leth; Lomdahl, P. S.

    1987-01-01

    In continuation of an earlier study of propagation of solitary waves on nonlinear elastic rods, numerical investigations of blowup, reflection, and fission at continuous and discontinuous variation of the cross section for the rod and reflection at the end of the rod are presented. The results...

  8. Phase behavior of colloidal silica rods

    NARCIS (Netherlands)

    Kuijk, A.; Byelov, D.; Petukhov, A.V.; van Blaaderen, A.; Imhof, A.

    2012-01-01

    Recently, a novel colloidal hard-rod-like model system was developed which consists of silica rods [Kuijk et al., JACS, 2011, 133, 2346]. Here, we present a study of the phase behavior of these rods, for aspect ratios ranging from 3.7 to 8.0. By combining real-space confocal laser scanning microscop

  9. Hydraulic Actuator for Ganged Control Rods

    Science.gov (United States)

    Thompson, D. C.; Robey, R. M.

    1986-01-01

    Hydraulic actuator moves several nuclear-reactor control rods in unison. Electromagnetic pump pushes liquid lithium against ends of control rods, forcing them out of or into nuclear reactor. Color arrows show lithium flow for reactor startup and operation. Flow reversed for shutdown. Conceived for use aboard spacecraft, actuator principle applied to terrestrial hydraulic machinery involving motion of ganged rods.

  10. Photocurrents of cone photoreceptors of the golden-mantled ground squirrel.

    Science.gov (United States)

    Kraft, T W

    1988-10-01

    1. Visual transduction in photoreceptors of the ground squirrel, Citellus lateralis, was studied by recording membrane current from individual cones in small pieces of retina. 2. Brief flashes of light produced transient reductions of the dark current; saturating response amplitudes were up to 67 pA. A flash strength of about 11,000 photons microns-2 at lambda max was required to give a half-saturating response. The stimulus-response relation was well fitted by an exponential saturation curve. Responses below 20% of maximum behaved linearly. 3. The response to a dim flash in most cells had a time to peak of 20-30 ms and resembled the impulse response of a series of five low-pass filters. 4. The variance of the dim-flash response amplitude put an upper limit of 80 fA on the size of the single photon response. Estimates based on the effective collecting area suggest the single photon response to be of the order of 10 fA. 5. Flash responses of squirrel cones usually lacked the undershoot observed in primate cones, although in about 1/3 of the cells a small undershoot developed during recording. 6. Background lights slightly shortened the time to peak of the flash response and reduced the integration time. 7. Spectral sensitivity measurements showed two classes of cones with peak sensitivities at about 520 and 435 nm. Rod sensitivity peaked near 500 nm. Spectral univariance was obeyed by all three classes of cells. 8. The shapes of the spectral sensitivity curves of the rod and both types of cones were similar to each other when plotted on a log wave number scale, but differed significantly from similar plots of monkey and human cone spectra. 9. The kinetics and sensitivity of flash responses of the blue- and green-sensitive cones were indistinguishable.

  11. Centrosomal-ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis.

    Science.gov (United States)

    Cideciyan, Artur V; Aleman, Tomas S; Jacobson, Samuel G; Khanna, Hemant; Sumaroka, Alexander; Aguirre, Geoffrey K; Schwartz, Sharon B; Windsor, Elizabeth A M; He, Shirley; Chang, Bo; Stone, Edwin M; Swaroop, Anand

    2007-11-01

    Mutations in the centrosomal-ciliary gene CEP290/NPHP6 are associated with Joubert syndrome and are the most common cause of the childhood recessive blindness known as Leber congenital amaurosis (LCA). An in-frame deletion in Cep290 shows rapid degeneration in the rod-rich mouse retina. To explore the mechanisms of the human retinal disease, we studied CEP290-LCA in patients of different ages (7-48 years) and compared results to Cep290-mutant mice. Unexpectedly, blind CEP290-mutant human retinas retained photoreceptor and inner laminar architecture in the cone-rich central retina, independent of severity of visual loss. Surrounding the cone-rich island was photoreceptor loss and distorted retina, suggesting neural-glial remodeling. The mutant mouse retina at 4-6 weeks of age showed similar features of retinal remodeling, with altered neural and synaptic laminae and Muller glial activation. The visual brain pathways in CEP290-LCA were anatomically intact. Our findings of preserved foveal cones and visual brain anatomy in LCA with CEP290 mutations, despite severe blindness and rapid rod cell death, suggest an opportunity for visual restoration of central vision in this common form of inherited blindness.

  12. Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin.

    Science.gov (United States)

    Wensel, T G; Stryer, L

    1986-09-01

    The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.

  13. ELECTROMAGNETIC APPARATUS FOR MOVING A ROD

    Science.gov (United States)

    Young, J.N.

    1958-04-22

    An electromagnetic apparatus for moving a rod-like member in small steps in either direction is described. The invention has particular application in the reactor field where the reactor control rods must be moved only a small distance and where the use of mechanical couplings is impractical due to the high- pressure seals required. A neutron-absorbing rod is mounted in a housing with gripping uaits that engage the rod, and coils for magnetizing the gripping units to make them grip, shift, and release the rod are located outside the housing.

  14. The hypothalamic photoreceptors regulating seasonal reproduction in birds: a prime role for VA opsin.

    Science.gov (United States)

    García-Fernández, José M; Cernuda-Cernuda, Rafael; Davies, Wayne I L; Rodgers, Jessica; Turton, Michael; Peirson, Stuart N; Follett, Brian K; Halford, Stephanie; Hughes, Steven; Hankins, Mark W; Foster, Russell G

    2015-04-01

    Extraretinal photoreceptors located within the medio-basal hypothalamus regulate the photoperiodic control of seasonal reproduction in birds. An action spectrum for this response describes an opsin photopigment with a λmax of ∼ 492 nm. Beyond this however, the specific identity of the photopigment remains unresolved. Several candidates have emerged including rod-opsin; melanopsin (OPN4); neuropsin (OPN5); and vertebrate ancient (VA) opsin. These contenders are evaluated against key criteria used routinely in photobiology to link orphan photopigments to specific biological responses. To date, only VA opsin can easily satisfy all criteria and we propose that this photopigment represents the prime candidate for encoding daylength and driving seasonal breeding in birds. We also show that VA opsin is co-expressed with both gonadotropin-releasing hormone (GnRH) and arginine-vasotocin (AVT) neurons. These new data suggest that GnRH and AVT neurosecretory pathways are endogenously photosensitive and that our current understanding of how these systems are regulated will require substantial revision.

  15. Exploiting rod technology. Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1990-06-01

    ROD development was proceeding apace until recent budgetary decisions caused funding support for ROD development to be drastically reduced. The funding which was originally provided by DARPA and the Balanced Technology Initiative (BTI) Office has been cut back to zero from $800K. To determine the aeroballistic coefficients of a candidate dart, ARDEC is currently supporting development out of its own 6.2 funds at about $100K. ARDEC has made slow progress toward achieving this end because of failures in the original dart during testing. It appears that the next dart design to be tested will diverge from the original concept visualized by DARPA and Science and Technology Associates (STA). STA, the design engineer, takes exception to these changes on the basis of inappropriate test conditions and insufficient testing. At this time, the full resolution of this issue will be difficult because of the current management structure, which separates the developer (ARDEC) from the designer (STA).

  16. Activation of autophagy in photoreceptor necroptosis after experimental retinal detachment

    Institute of Scientific and Technical Information of China (English)

    Kai; Dong; Zi-Cheng; Zhu; Feng-Hua; Wang; Gen-Jie; Ke; Zhang; Yu; Xun; Xu

    2014-01-01

    AIM:To investigate whether photoreceptor necroptosis induced by z-VAD-FMK(pan caspase inhibitor) was involved the activation of autophagy and whether Necrostatin-1, a specific necroptosis inhibitor, could inhibit this induction of autophagy after experimental retinal detachment.METHODS:Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate and subretinal injections of z-VAD-FMK, vehicle or z-VAD-FMK plus Necrostatin-1.Three days after retinal detachment, morphologic changes were observed by transmission electron microscopy. In other animals, retinas were subjected to immunoprecipitation and Western Blotting, then probed with anti-RIP1, phosphoserine, LC-3II or caspase 8antibody.RESULTS:It was proved by immunoprecipitation and western blotting, that photoreceptor necroptosis was mediated by caspase-8 inhibition and receptor interacting protein kinase(RIP1) phosphorylation activation. Transmission electron microscope and western blotting results indicated that photoreceptornecroptosis was involved the LC-3II and autophagosomes induction. We also discovered Necrostatin-1 could inhibit RIP1 phosphorylation and LC-3II induction.CONCLUSION:These data firstly indicate photoreceptor necroptosis is associated with the activation of autophagy. Necrostatin-1 protects photoreceptors from necroptosis and autophagy by down-regulation of RIP1 phosphorylation and LC-3II.

  17. Waveguide Modes and Refractive Index in Photoreceptors of Invertebrates

    NARCIS (Netherlands)

    Stavenga, D.G.

    1975-01-01

    The refractive index of visual photoreceptors, if estimated by utilizing waveguide propagation, has to be corrected by a factor depending on the occurring mode. The correction factor is presented graphically for a number of relevant modes. Applied to the honeybee rhabdoms, it is shown that the

  18. Angular sensitivity of blowfly photoreceptors : broadening by artificial electrical coupling

    NARCIS (Netherlands)

    Smakman, J.G.J.; Stavenga, D.G.

    1987-01-01

    1. Electrical coupling between R1-6 photoreceptors was investigated by measuring angular sensitivities and quantum bumps. 2. Recordings were made from two extreme types of cells: Type a: cells with a diffraction-like angular sensitivity profile. Only large bumps could be obtained from these cells. T

  19. Dlic1 deficiency impairs ciliogenesis of photoreceptors by destabilizing dynein

    Institute of Scientific and Technical Information of China (English)

    Shanshan Kong; Xinrong Du; Chao Peng; Yiming Wu; Huirong Li; Xi Jin; Ling Hou

    2013-01-01

    Cytoplasmic dynein 1 is fundamentally important for transporting a variety of essential cargoes along microtubules within eukaryotic cells.However,in mammals,few mutants are available for studying the effects of defects in dynein-controlled processes in the context of the whole organism.Here,we deleted mouse Dlic1 gene encoding DLIC1,a subunit of the dynein complex.Dlic1-/-mice are viable,but display severe photoreceptor degeneration.Ablation of Dlic1 results in ectopic accumulation of outer segment (OS) proteins,and impairs OS growth and ciliogenesis of photoreceptors by interfering with Rabll-vesicle trafficking and blocking efficient OS protein transport from Golgi to the basal body.Our studies show that Dlic1 deficiency partially blocks vesicle export from endoplasmic reticulum (ER),but seems not to affect vesicle transport from the ER to Golgi.Further mechanistic study reveals that lack of Dlic1 destabilizes dynein subunits and alters the normal subcellular distribution of dynein in photoreceptors,probably due to the impaired transport function of dynein.Our results demonstrate that Dlic1 plays important roles in ciliogenesis and protein transport to the OS,and is required for photoreceptor development and survival.The Dlic1-/-mice also provide a new mouse model to study human retinal degeneration.

  20. Diurnal Changes in Angular Sensitivity of Crab Photoreceptors

    NARCIS (Netherlands)

    Leggett, L.M.W.; Stavenga, D.G.

    1981-01-01

    The electrophysiological and anatomical consequences of diurnal changes in screening pigment position were investigated in the apposition eye of the portunid crab Scylla serrata. Intracellular recordings revealed that the acceptance angles of dark-adapted photoreceptors enlarged up to four-fold at n

  1. Three spectrally distinct photoreceptors in diurnal and nocturnal Australian ants.

    Science.gov (United States)

    Ogawa, Yuri; Falkowski, Marcin; Narendra, Ajay; Zeil, Jochen; Hemmi, Jan M

    2015-06-07

    Ants are thought to be special among Hymenopterans in having only dichromatic colour vision based on two spectrally distinct photoreceptors. Many ants are highly visual animals, however, and use vision extensively for navigation. We show here that two congeneric day- and night-active Australian ants have three spectrally distinct photoreceptor types, potentially supporting trichromatic colour vision. Electroretinogram recordings show the presence of three spectral sensitivities with peaks (λmax) at 370, 450 and 550 nm in the night-active Myrmecia vindex and peaks at 370, 470 and 510 nm in the day-active Myrmecia croslandi. Intracellular electrophysiology on individual photoreceptors confirmed that the night-active M. vindex has three spectral sensitivities with peaks (λmax) at 370, 430 and 550 nm. A large number of the intracellular recordings in the night-active M. vindex show unusually broad-band spectral sensitivities, suggesting that photoreceptors may be coupled. Spectral measurements at different temporal frequencies revealed that the ultraviolet receptors are comparatively slow. We discuss the adaptive significance and the probability of trichromacy in Myrmecia ants in the context of dim light vision and visual navigation. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  2. CNGA3 mutations in hereditary cone photoreceptor disorders

    NARCIS (Netherlands)

    Wissinger, B; Gamer, D; Jagle, H; Giorda, R; Marx, T; Mayer, S; Tippmann, S; Broghammer, M; Jurklies, B; Rosenberg, T; Jacobson, SG; Sener, EC; Tatlipinar, S; Hoyng, CB; Castellan, C; Bitoun, P; Andreasson, S; Rudolph, G; Kellner, U; Lorenz, B; Wolff, G; Verellen-Dumoulin, C; Schwartz, M; Cremers, FPM; Apfelstedt-ylla, E; Zrenner, E; Salati, R; Sharpe, LT; Kohl, S

    2001-01-01

    We recently showed that mutations in the CNGA3 gene encoding the alpha -subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258

  3. Diurnal Changes in Angular Sensitivity of Crab Photoreceptors

    NARCIS (Netherlands)

    Leggett, L.M.W.; Stavenga, D.G.

    1981-01-01

    The electrophysiological and anatomical consequences of diurnal changes in screening pigment position were investigated in the apposition eye of the portunid crab Scylla serrata. Intracellular recordings revealed that the acceptance angles of dark-adapted photoreceptors enlarged up to four-fold at

  4. Chloroplasts continuously monitor photoreceptor signals during accumulation movement.

    Science.gov (United States)

    Tsuboi, Hidenori; Wada, Masamitsu

    2013-07-01

    Under low light conditions, chloroplasts gather at a cell surface to maximize light absorption for efficient photosynthesis, which is called the accumulation response. Phototropin1 (phot1) and phototropin2 (phot2) were identified as blue light photoreceptors in the accumulation response that occurs in Arabidopsis thaliana and Adiantum capillus-veneris with neochrome1 (neo1) as a red light photoreceptor in A. capillus-veneris. However, the signal molecule that is emitted from the photoreceptors and transmitted to the chloroplasts is not known. To investigate this topic, the accumulation response was induced by partial cell irradiation with a microbeam of red, blue and far-red light in A. capillus-veneris gametophyte cells. Chloroplasts moved towards the irradiated region and were able to sense the signal as long as its signal flowed. The signal from neo1 had a longer life than the signal that came from phototropins. When two microbeams with the same wavelength and the same fluence rate were placed 20 μm apart from each other and were applied to a dark-adapted cell, chloroplasts at an equidistant position always moved towards the center (midpoint) of the two microbeams, but not towards either one. This result indicates that chloroplasts are detecting the concentration of the signal but not the direction of signal flow. Chloroplasts repeatedly move and stop at roughly 10 s intervals during the accumulation response, suggesting that they monitor the intermittent signal waves from photoreceptors.

  5. CNGA3 mutations in hereditary cone photoreceptor disorders

    NARCIS (Netherlands)

    Wissinger, B; Gamer, D; Jagle, H; Giorda, R; Marx, T; Mayer, S; Tippmann, S; Broghammer, M; Jurklies, B; Rosenberg, T; Jacobson, SG; Sener, EC; Tatlipinar, S; Hoyng, CB; Castellan, C; Bitoun, P; Andreasson, S; Rudolph, G; Kellner, U; Lorenz, B; Wolff, G; Verellen-Dumoulin, C; Schwartz, M; Cremers, FPM; Apfelstedt-ylla, E; Zrenner, E; Salati, R; Sharpe, LT; Kohl, S

    2001-01-01

    We recently showed that mutations in the CNGA3 gene encoding the alpha -subunit of the cone photoreceptor cGMP-gated channel cause autosomal recessive complete achromatopsia linked to chromosome 2q11. We now report the results of a first comprehensive screening for CNGA3 mutations in a cohort of 258

  6. CSF concentrations of cAMP and cGMP are lower in patients with Creutzfeldt-Jakob disease but not Parkinson's disease and amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Patrick Oeckl

    Full Text Available BACKGROUND: The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP and cyclic guanosine-3',5'-monophosphate (cGMP are important second messengers and are potential biomarkers for Parkinson's disease (PD, amyotrophic lateral sclerosis (ALS and Creutzfeldt-Jakob disease (CJD. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS the cerebrospinal fluid (CSF concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15 had lower cAMP (-70% and cGMP (-55% concentrations in CSF compared with controls (n = 11. There was no difference in PD, PD dementia (PDD and ALS cases. Receiver operating characteristic (ROC curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC of 0.86 (cAMP and 0.85 (cGMP. We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92 and to 93% and 100% for the ratio tau/cAMP (AUC 0.99. CONCLUSIONS/SIGNIFICANCE: We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control.

  7. Retinal Thickening and Photoreceptor Loss in HIV Eyes without Retinitis.

    Directory of Open Access Journals (Sweden)

    Cheryl A Arcinue

    Full Text Available To determine the presence of structural changes in HIV retinae (i.e., photoreceptor density and retinal thickness in the macula compared with age-matched HIV-negative controls.Cohort of patients with known HIV under CART (combination Antiretroviral Therapy treatment were examined with a flood-illuminated retinal AO camera to assess the cone photoreceptor mosaic and spectral-domain optical coherence tomography (SD-OCT to assess retinal layers and retinal thickness.Twenty-four eyes of 12 patients (n = 6 HIV-positive and 6 HIV-negative were imaged with the adaptive optics camera. In each of the regions of interest studied (nasal, temporal, superior, inferior, the HIV group had significantly less mean cone photoreceptor density compared with age-matched controls (difference range, 4,308-6,872 cones/mm2. A different subset of forty eyes of 20 patients (n = 10 HIV-positive and 10 HIV-negative was included in the retinal thickness measurements and retinal layer segmentation with the SD-OCT. We observed significant thickening in HIV positive eyes in the total retinal thickness at the foveal center, and in each of the three horizontal B-scans (through the macular center, superior, and inferior to the fovea. We also noted that the inner retina (combined thickness from ILM through RNFL to GCL layer was also significantly thickened in all the different locations scanned compared with HIV-negative controls.Our present study shows that the cone photoreceptor density is significantly reduced in HIV retinae compared with age-matched controls. HIV retinae also have increased macular retinal thickness that may be caused by inner retinal edema secondary to retinovascular disease in HIV. The interaction of photoreceptors with the aging RPE, as well as possible low-grade ocular inflammation causing diffuse inner retinal edema, may be the key to the progressive vision changes in HIV-positive patients without overt retinitis.

  8. Restoration of Vision in the pde6β-deficient Dog, a Large Animal Model of Rod-cone Dystrophy

    Science.gov (United States)

    Petit, Lolita; Lhériteau, Elsa; Weber, Michel; Le Meur, Guylène; Deschamps, Jack-Yves; Provost, Nathalie; Mendes-Madeira, Alexandra; Libeau, Lyse; Guihal, Caroline; Colle, Marie-Anne; Moullier, Philippe; Rolling, Fabienne

    2012-01-01

    Defects in the β subunit of rod cGMP phosphodiesterase 6 (PDE6β) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6β (n = 4) or AAV2/8RK.cpde6β (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6β- and AAV2/8RK.cpde6β-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment. PMID:22828504

  9. The Role of Aquaporin 1 Activated by cGMP in Myocardial Edema Caused by Cardiopulmonary Bypass in Sheep

    Directory of Open Access Journals (Sweden)

    Fang-bao Ding

    2013-11-01

    Full Text Available Background/Aims: Most cardiac procedures involve the use of cardiopulmonary bypass (CPB, which pumps oxygenated blood to the body while the heart and lungs are isolated. CPB can cause profound alterations V in the homeostasis of physiological fluids, which often results in myocardial edema. In our study, we used sheep CPB model of in vivo and in vitro to assess the relationship between cGMP and AQP1 during CPB. Methods: ODQ, a specific inhibitor of soluble guanylate cyclase (sGC, was used to treat the CPB animals or cardiomyocytes. Left ventricular function of each group was determined by pressure-volume system. Water content of myocardial tissue was assessed by dry-wet weight, and cardiomyocytes water permeability was also calculated. The concentration of cGMP was determined by Radioimmunoassay (RIA. mRNA and protein expression of AQP1 were detected by real-time PCR and western blot, respectively. Results: The relative expression level of AQP1 mRNA and protein at each time point (0, 6, 12, 24 or 48 h after CPB was significantly increased (1.18-fold at 12 h, 1.77-fold at 24 h and 2.18-fold at 48h compared with each sham group, the protein expression of AQP1 also showed a rising trend after CPB. The degree of myocardial edema (75.1% at 12 h, 79.3% at 24 h and 81.0% at 48h increased following the CPB surgery. The mRNA expression level of AQP1 was significantly decreased by 39.7% (pin vitro experiments showed the same changing trends as in vivo. Conclusion: cGMP pathway controls water channels and then affects water intake during CPB through an AQP1-mediated pathway.

  10. The Importance of cGMP Signaling in Sensory Cilia for Body Size Regulation in Caenorhabditis elegans.

    Science.gov (United States)

    Fujiwara, Manabi; Hino, Takahiro; Miyamoto, Ryuta; Inada, Hitoshi; Mori, Ikue; Koga, Makoto; Miyahara, Koji; Ohshima, Yasumi; Ishihara, Takeshi

    2015-12-01

    The body size of Caenorhabditis elegans is thought to be controlled by sensory inputs because many mutants with sensory cilium structure defects exhibit small body size. The EGL-4 cGMP-dependent protein kinase acts in sensory neurons to reduce body size when animals fail to perceive sensory signals. In addition to body size control, EGL-4 regulates various other behavioral and developmental pathways, including those involved in the regulation of egg laying and chemotaxis behavior. Here we have identified gcy-12, which encodes a receptor-type guanylyl cyclase, as a gene involved in the sensory regulation of body size. Analyses with GFP fusion constructs showed that gcy-12 is expressed in several sensory neurons and localizes to sensory cilia. Genetic analyses indicated that GCY-12 acts upstream of EGL-4 in body size control but does not affect other EGL-4 functions. Our studies indicate that the function of the GCY-12 guanylyl cyclase is to provide cGMP to the EGL-4 cGMP-dependent kinase only for limited tasks including body size regulation. We also found that the PDE-2 cyclic nucleotide phosphodiesterase negatively regulates EGL-4 in controlling body size. Thus, the cGMP level is precisely controlled by GCY-12 and PDE-2 to determine body size through EGL-4, and the defects in the sensory cilium structure may disturb the balanced control of the cGMP level. The large number of guanylyl cyclases encoded in the C. elegans genome suggests that EGL-4 exerts pleiotropic effects by partnering with different guanylyl cyclases for different downstream functions.

  11. The phototransduction machinery in the rod outer segment has a strong efficacy gradient

    KAUST Repository

    Mazzolini, Monica

    2015-05-04

    Rod photoreceptors consist of an outer segment (OS) and an inner segment. Inside the OS a biochemical machinery transforms the rhodopsin photoisomerization into electrical signal. This machinery has been treated as and is thought to be homogenous with marginal inhomogeneities. To verify this assumption, we developed a methodology based on special tapered optical fibers (TOFs) to deliver highly localized light stimulations. By using these TOFs, specific regions of the rod OS could be stimulated with spots of light highly confined in space. As the TOF is moved from the OS base toward its tip, the amplitude of saturating and single photon responses decreases, demonstrating that the efficacy of the transduction machinery is not uniform and is 5-10 times higher at the base than at the tip. This gradient of efficacy of the transduction machinery is attributed to a progressive depletion of the phosphodiesterase along the rod OS. Moreover we demonstrate that, using restricted spots of light, the duration of the photoresponse along the OS does not increase linearly with the light intensity as with diffuse light. © 2015, National Academy of Sciences. All rights reserved.

  12. The phototransduction machinery in the rod outer segment has a strong efficacy gradient.

    Science.gov (United States)

    Mazzolini, Monica; Facchetti, Giuseppe; Andolfi, Laura; Proietti Zaccaria, Remo; Tuccio, Salvatore; Treu, Johannes; Altafini, Claudio; Di Fabrizio, Enzo M; Lazzarino, Marco; Rapp, Gert; Torre, Vincent

    2015-05-19

    Rod photoreceptors consist of an outer segment (OS) and an inner segment. Inside the OS a biochemical machinery transforms the rhodopsin photoisomerization into electrical signal. This machinery has been treated as and is thought to be homogenous with marginal inhomogeneities. To verify this assumption, we developed a methodology based on special tapered optical fibers (TOFs) to deliver highly localized light stimulations. By using these TOFs, specific regions of the rod OS could be stimulated with spots of light highly confined in space. As the TOF is moved from the OS base toward its tip, the amplitude of saturating and single photon responses decreases, demonstrating that the efficacy of the transduction machinery is not uniform and is 5-10 times higher at the base than at the tip. This gradient of efficacy of the transduction machinery is attributed to a progressive depletion of the phosphodiesterase along the rod OS. Moreover we demonstrate that, using restricted spots of light, the duration of the photoresponse along the OS does not increase linearly with the light intensity as with diffuse light.

  13. Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells.

    Science.gov (United States)

    Li, Tianqing; Lewallen, Michelle; Chen, Shuyi; Yu, Wei; Zhang, Nian; Xie, Ting

    2013-06-01

    Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptor-deficient mice, but there is still some concern of tumor formation. In this study, we have successfully cultured Nestin(+)Sox2(+)Pax6(+) multipotent retinal stem cells (RSCs) from the adult mouse retina, which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation. After they have been expanded for over 35 passages in the presence of FGF and EGF, the cultured RSCs still maintain stable proliferation and differentiation potential. Under proper differentiation conditions, they can differentiate into all the major retinal cell types found in the adult retina. More importantly, they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions. Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes, RSC-derived photoreceptor cells integrate into the retina, morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons. When transplanted into eyes of photoreceptor-deficient rd1 mutant mice, a RP model, RSC-derived photoreceptors can partially restore light response, indicating that those RSC-derived photoreceptors are functional. Finally, there is no evidence for tumor formation in the photoreceptor-transplanted eyes. Therefore, this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD.

  14. The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling

    CSIR Research Space (South Africa)

    Wheeler, J

    2017-06-01

    Full Text Available . Data were analysed by two-way ANOVA followed by Sidak’s multiple comparisons post-hoc test. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd, The Plant Journal, (2017), doi: 10.1111/tpj.13589 BRI1 generates cGMP to modulate....49 � 0.132 pmol/lg protein (Figure 1c). A BRI model incorporating the KR1083/4AA mutations was able to form a helical structural fold (Fig- ure S1) that characterizes all Arabidopsis GC centres reported to-date (Wong et al., 2015) and thus may account...

  15. Raising the standard: changes to the Australian Code of Good Manufacturing Practice (cGMP) for human blood and blood components, human tissues and human cellular therapy products.

    Science.gov (United States)

    Wright, Craig; Velickovic, Zlatibor; Brown, Ross; Larsen, Stephen; Macpherson, Janet L; Gibson, John; Rasko, John E J

    2014-04-01

    In Australia, manufacture of blood, tissues and biologicals must comply with the federal laws and meet the requirements of the Therapeutic Goods Administration (TGA) Manufacturing Principles as outlined in the current Code of Good Manufacturing Practice (cGMP). The Therapeutic Goods Order (TGO) No. 88 was announced concurrently with the new cGMP, as a new standard for therapeutic goods. This order constitutes a minimum standard for human blood, tissues and cellular therapeutic goods aimed at minimising the risk of infectious disease transmission. The order sets out specific requirements relating to donor selection, donor testing and minimisation of infectious disease transmission from collection and manufacture of these products. The Therapeutic Goods Manufacturing Principles Determination No. 1 of 2013 references the human blood and blood components, human tissues and human cellular therapy products 2013 (2013 cGMP). The name change for the 2013 cGMP has allowed a broadening of the scope of products to include human cellular therapy products. It is difficult to directly compare versions of the code as deletion of some clauses has not changed the requirements to be met, as they are found elsewhere amongst the various guidelines provided. Many sections that were specific for blood and blood components are now less prescriptive and apply to a wider range of cellular therapies, but the general overall intent remains the same. Use of 'should' throughout the document instead of 'must' allows flexibility for alternative processes, but these systems will still require justification by relevant logical argument and validation data to be acceptable to TGA. The cGMP has seemingly evolved so that specific issues identified at audit over the last decade have now been formalised in the new version. There is a notable risk management approach applied to most areas that refer to process justification and decision making. These requirements commenced on 31 May 2013 and a 12 month

  16. The Receptor-Bound Guanylyl Cyclase DAF-11 Is the Mediator of Hydrogen Peroxide-Induced cGMP Increase in Caenorhabditis elegans [corrected]..

    Directory of Open Access Journals (Sweden)

    Ulrike Beckert

    Full Text Available Adenosine 3', 5'-cyclic monophosphate (cAMP and guanosine 3', 5'-cyclic monophosphate (cGMP are well-studied second messengers that transmit extracellular signals into mammalian cells, with conserved functions in various other species such as Caenorhabditis elegans (C. elegans. cAMP is generated by adenylyl cyclases, and cGMP is generated by guanylyl cyclases, respectively. Studies using C. elegans have revealed additional roles for cGMP signaling in lifespan extension. For example, mutants lacking the function of a specific receptor-bound guanylyl cyclase, DAF-11, have an increased life expectancy. While the daf-11 phenotype has been attributed to reductions in intracellular cGMP concentrations, the actual content of cyclic nucleotides has not been biochemically determined in this system. Similar assumptions were made in studies using phosphodiesterase loss-of-function mutants or using adenylyl cyclase overexpressing mutants. In the present study, cyclic nucleotide regulation in C. elegans was studied by establishing a special nematode protocol for the simultaneous detection and quantitation of cyclic nucleotides. We also examined the influence of reactive oxygen species (ROS on cyclic nucleotide metabolism and lifespan in C. elegans using highly specific HPLC-coupled tandem mass-spectrometry and behavioral assays. Here, we show that the relation between cGMP and survival is more complex than previously appreciated.

  17. High temperature control rod assembly

    Energy Technology Data Exchange (ETDEWEB)

    Vollman, Russell E. (Solana Beach, CA)

    1991-01-01

    A high temperature nuclear control rod assembly comprises a plurality of substantially cylindrical segments flexibly joined together in succession by ball joints. The segments are made of a high temperature graphite or carbon-carbon composite. The segment includes a hollow cylindrical sleeve which has an opening for receiving neutron-absorbing material in the form of pellets or compacted rings. The sleeve has a threaded sleeve bore and outer threaded surface. A cylindrical support post has a threaded shaft at one end which is threadably engaged with the sleeve bore to rigidly couple the support post to the sleeve. The other end of the post is formed with a ball portion. A hollow cylindrical collar has an inner threaded surface engageable with the outer threaded surface of the sleeve to rigidly couple the collar to the sleeve. the collar also has a socket portion which cooperates with the ball portion to flexibly connect segments together to form a ball and socket-type joint. In another embodiment, the segment comprises a support member which has a threaded shaft portion and a ball surface portion. The threaded shaft portion is engageable with an inner threaded surface of a ring for rigidly coupling the support member to the ring. The ring in turn has an outer surface at one end which is threadably engageably with a hollow cylindrical sleeve. The other end of the sleeve is formed with a socket portion for engagement with a ball portion of the support member. In yet another embodiment, a secondary rod is slidably inserted in a hollow channel through the center of the segment to provide additional strength. A method for controlling a nuclear reactor utilizing the control rod assembly is also included.

  18. Topological Mixing with Ghost Rods

    OpenAIRE

    2005-01-01

    Topological chaos relies on the periodic motion of obstacles in a two-dimensional flow in order to form nontrivial braids. This motion generates exponential stretching of material lines, and hence efficient mixing. Boyland et al. [P. L. Boyland, H. Aref, and M. A. Stremler, J. Fluid Mech. 403, 277 (2000)] have studied a specific periodic motion of rods that exhibits topological chaos in a viscous fluid. We show that it is possible to extend their work to cases where the motion of the stirring...

  19. Reactor control rod timing system. [LMFBR

    Science.gov (United States)

    Wu, P.T.K.

    1980-03-18

    A fluid driven jet-edge whistle timing system is described for control rods of a nuclear reactor for producing real-time detection of the timing of each control rod in its scram operation. An important parameter in reactor safety, particularly for liquid metal fast breeder reactors (LMFBR), is the time deviation between the time the control rod is released and the time the rod actually reaches the down position. The whistle has a nearly pure tone signal with center frequency (above 100 kHz) far above the frequency band in which the energy of the background noise is concentrated. Each control rod can be fitted with a whistle with a different frequency so that there is no ambiguity in differentiating the signal from each control rod.

  20. The Retinal Pigment Epithelium: a Convenient Source of New Photoreceptor cells?

    Directory of Open Access Journals (Sweden)

    Shu-Zhen Wang

    2014-01-01

    Full Text Available Recent success in restoring visual function through photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to generate or regenerate photoreceptor cells for the ultimate goal of using cell replacement therapy for blindness caused by photoreceptor degeneration. Current research on deriving new photoreceptors for replacement, as regenerative medicine in general, focuses on the use of embryonic stem cells and induced pluripotent stem (iPS cells to generate transplantable cells. Nonetheless, naturally occurring regeneration, such as wound healing, involves awakening cells at or near a wound site to produce new cells needed to heal the wound. Here we discuss the possibility of tweaking an ocular tissue, the retinal pigment epithelium (RPE, to produce photoreceptor cells in situ in the eye. Unlike the neural retina, the RPE in adult mammals maintains cell proliferation capability. Furthermore, progeny cells from RPE proliferation may differentiate into cells other than RPE. The combination of proliferation and plasticity opens a question of whether they could be channeled by a regulatory gene with pro-photoreceptor activity towards photoreceptor production. Studies using embryonic chick and transgenic mouse showed that indeed photoreceptor-like cells were produced in culture and in vivo in the eye using genedirected reprogramming of RPE cells, supporting the feasibility of using the RPE as a convenient source of new photoreceptor cells for in situ retinal repair without involving cell transplantation.

  1. Control of a four-color sensing photoreceptor by a two-color sensing photoreceptor reveals complex light regulation in cyanobacteria

    OpenAIRE

    Bussell, Adam N.; Kehoe, David M.

    2013-01-01

    Photoreceptors are biologically important for sensing changes in the color and intensity of ambient light and, for photosynthetic organisms, processing this light information to optimize food production through photosynthesis. Cyanobacteria are an evolutionarily and ecologically important prokaryotic group of oxygenic photosynthesizers that contain cyanobacteriochrome (CBCR) photoreceptors, whose family members sense nearly the entire visible spectrum of light colors. Some cyanobacteria conta...

  2. Automatic safety rod for reactors. [LMFBR

    Science.gov (United States)

    Germer, J.H.

    1982-03-23

    An automatic safety rod for a nuclear reactor containing neutron absorbing material and designed to be inserted into a reactor core after a loss-of-flow. Actuation is based upon either a sudden decrease in core pressure drop or the pressure drop decreases below a predetermined minimum value. The automatic control rod includes a pressure regulating device whereby a controlled decrease in operating pressure due to reduced coolant flow does not cause the rod to drop into the core.

  3. Carbenoxolone blocks the light-evoked rise in intracellular calcium in isolated melanopsin ganglion cell photoreceptors.

    Directory of Open Access Journals (Sweden)

    Jayne R Bramley

    Full Text Available BACKGROUND: Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs. These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca(2+ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca(2+ signals in ipRGCs independent of gap junction blockade. METHODOLOGY/PRINCIPAL FINDINGS: To test the possibility that carbenoxolone directly inhibits light-evoked Ca(2+ responses in ipRGCs, the light-evoked rise in intracellular Ca(2+ ([Ca(2+](i was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca(2+](i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the light-evoked rise in [Ca(2+](i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca(2+](i in isolated ipRGCs is almost entirely due to Ca(2+ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca(2+](i in ipRGCs by blocking L-type voltage-gated Ca(2+ channels. The ability of

  4. Hydralazine decreases sodium nitroprusside-induced rat aortic ring relaxation and increased cGMP production by rat aortic myocytes.

    Science.gov (United States)

    Vidrio, Horacio; González-Romo, Pilar; Alvarez, Ezequiel; Alcaide, Carlos; Orallo, Francisco

    2005-10-28

    Association of hydralazine with nitrova-sodilators has long been known to be beneficial in the vasodilator treatment of heart failure. We previously found that hydralazine appeared to reduce the increase in cGMP induced by sodium nitroprusside in cultured rat aortic myocytes. In order to further explore this seemingly paradoxical interaction, we extended our initial observations in rat aortic myocytes and also determined the influence of hydralazine on sodium nitroprusside-induced relaxation of rat aortic rings. Hydralazine produced a concentration-dependent inhibition of sodium nitroprusside stimulation of cGMP production and caused a rightward shift of concentration-relaxation curves in aortic rings. A possible mechanism of the hydralazine-nitroprusside interaction could be the interference with bioactivation of the nitro-vasodilator to release nitric oxide. Recent evidence indicates that vascular NADH oxidase, an enzyme known to be inhibited by hydralazine, could be involved in this process. Accordingly, hydralazine was found to inhibit NADH oxidase activity in rat aortic myocytes at concentrations similar to those reducing sodium nitroprusside responses. It was concluded that antagonism of sodium nitroprusside action by hydralazine could be a consequence of interference with bioactivation of the former, apparently through inhibition of vascular NADH oxidase.

  5. Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells

    Institute of Scientific and Technical Information of China (English)

    Tianqing Li; Michelle Lewallen; Shuyi Chen; Wei Yu; Nian Zhang; Ting Xie

    2013-01-01

    Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases,such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD).Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptordeficient mice,but there is still some concern of tumor formation.In this study,we have successfully cultured Nestin+Sox2+Pax6+ multipotent retinal stem cells (RSCs) from the adult mouse retina,which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation.After they have been expanded for over 35 passages in the presence of FGF and EGF,the cultured RSCs still maintain stable proliferation and differentiation potential.Under proper differentiation conditions,they can differentiate into all the major retinal cell types found in the adult retina.More importantly,they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions.Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes,RSC-derived photoreceptor cells integrate into the retina,morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons.When transplanted into eyes of photoreceptor-deficient rd1 mutant mice,a RP model,RSC-derived photoreceptors can partially restore light response,indicating that those RSC-derived photoreceptors are functional.Finally,there is no evidence for tumor formation in the photoreceptor-transplanted eyes.Therefore,this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD.

  6. Potentiation of cGMP signaling increases oxygen delivery and oxidative metabolism in contracting skeletal muscle of older but not young humans

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Piil, Peter Bergmann; Egelund, Jon

    2015-01-01

    to evaluate the effect of inhibiting the main enzyme involved in cGMP degradation, phosphodiesterase 5 (PDE5), on blood flow and O2 delivery in contracting skeletal muscle of young and older humans. A group of young (23 ± 1 years) and a group of older (72 ± 2 years) male human subjects performed submaximal......Aging is associated with progressive loss of cardiovascular and skeletal muscle function. The impairment in physical capacity with advancing age could be related to an insufficient peripheral O2 delivery to the exercising muscles. Furthermore, the mechanisms underlying an impaired blood flow...... regulation remain unresolved. Cyclic guanosine monophosphate (cGMP) is one of the main second messengers that mediate smooth muscle vasodilation and alterations in cGMP signaling could, therefore, be one mechanism by which skeletal muscle perfusion is impaired with advancing age. The current study aimed...

  7. Acoustic loading effects on oscillating rod bundles

    Energy Technology Data Exchange (ETDEWEB)

    Lin, W.H.

    1980-01-01

    An analytical study of the interaction between an infinite acoustic medium and a cluster of circular rods is described. The acoustic field due to oscillating rods and the acoustic loading on the rods are first solved in a closed form. The acoustic loading is then used as a forcing function for rod responses, and the acousto-elastic couplings are solved simultaneously. Numerical examples are presented for several cases to illustrate the effects of various system parameters on the acoustic reaction force coefficients. The effect of the acoustic loading on the coupled eigenfrequencies are discussed.

  8. Discussion on Photoreceptor for Negative Phototropism in Rice Roots

    Institute of Scientific and Technical Information of China (English)

    WANG Yue-xia; WANG Zhong; SUO Biao; GU Yun-jie; WANG Hui-hui; CHEN Yong-hui; DAI Yun-xia

    2007-01-01

    To properly explore the photoreceptor for the negative phototropism in rice (Oryza sativa L.) root, lights with different wavelengths were applied to investigate the effect of light quality on phototropic bending. The phototropic bending could be induced prominently by blue/ultraviolet light, whereas not by red or far-red light. The absorption spectrum of the extracted solution from rice root cap had two peaks at 350 nm and 450 nm, respectively, and the molecular weight of the 120 kD protein in the root cap under unilateral light was larger than that under the dark. It suggested that the blue light receptor might be the photoreceptor for the negative phototropism in rice root.

  9. Discussion on Photoreceptor for Negative Phototropism in Rice Roots

    Directory of Open Access Journals (Sweden)

    Yue-xia WANG

    2007-12-01

    Full Text Available To properly explore the photoreceptor for the negative phototropism in rice (Oryza sativa L. root, lights with different wavelengths were applied to investigate the effect of light quality on phototropic bending. The phototropic bending could be induced prominently by blue/ultraviolet light, whereas not by red or far-red light. The absorption spectrum of the extracted solution from rice root cap had two peaks at 350 nm and 450 nm, respectively, and the molecular weight of the 120 kD protein in the root cap under unilateral light was larger than that under the dark. It suggested that the blue light receptor might be the photoreceptor for the negative phototropism in rice root.

  10. Amyloid precursor protein is required for normal function of the rod and cone pathways in the mouse retina.

    Directory of Open Access Journals (Sweden)

    Tracy Ho

    Full Text Available Amyloid precursor protein (APP is a transmembrane glycoprotein frequently studied for its role in Alzheimer's disease. Our recent study in APP knockout (KO mice identified an important role for APP in modulating normal neuronal development in the retina. However the role APP plays in the adult retina and whether it is required for vision is unknown. In this study we evaluated the role of APP in retinal function and morphology comparing adult wildtype (WT and APP-KO mice. APP was expressed on neuronal cells of the inner retina, including horizontal, cone bipolar, amacrine and ganglion cells in WT mice. The function of the retina was assessed using the electroretinogram and although the rod photoreceptor responses were similar in APP-KO and WT mice, the post-photoreceptor, inner retinal responses of both the rod and cone pathways were reduced in APP-KO mice. These changes in inner retinal function did not translate to a substantial change in visual acuity as assessed using the optokinetic response or to changes in the gross cellular structure of the retina. These findings indicate that APP is not required for basic visual function, but that it is involved in modulating inner retinal circuitry.

  11. Nr2e3 and Nrl can reprogram retinal precursors to the rod fate in Xenopus retina.

    Science.gov (United States)

    McIlvain, Vera A; Knox, Barry E

    2007-07-01

    Transformation of undifferentiated progenitors into specific cell types is largely dependent on temporal and spatial expression of a complex network of transcription factors. Here, we examined whether neural retina leucine zipper (Nrl) and photoreceptor-specific nuclear receptor Nr2e3 transcription factors contribute to cell fate determination. We cloned the Xenopus Nr2e3 gene and showed that its temporal and spatial expression is similar to its mammalian ortholog. We tested its in vivo function by misexpressing these transcription factors in Xenopus eye primordia, demonstrating that either human Nr2e3 or Nrl directed photoreceptor precursors to become rods at the expense of cones. Furthermore, overexpression of Xenopus Nrl dramatically increased the number of lens fibers, whereas human Nrl did not, suggesting evolutionary divergence of function of the Nrl gene family. Misexpression of Nrl and Nr2e3 together were more effective than either transcription factor alone in directing precursors to the rod fate. Copyright 2007 Wiley-Liss, Inc.

  12. Calcium pump in the disk membranes isolated from bovine retinal rod outer segments.

    Science.gov (United States)

    Panfoli, I; Morelli, A; Pepe, I M

    1994-08-01

    The existence of a Ca2+ pump in rod outer segment disks of bovine retina is strongly suggested by the isolation on sodium dodecyl sulfate polyacrylamide gel electrophoresis of a hydroxylamine-sensitive phosphorylated intermediate (E-P) of molecular mass of about 100 kDa as well as by measurements of active calcium transport and adenosine 5'-triphosphate (ATP) hydrolysis. Active Ca2+ uptake by disks was dependent on the presence of Mg(2+)-ATP, was inhibited by vanadate or lanthanum and appeared poorly sensitive to calmodulin. ATP hydrolysis by disk membranes was a function of free Ca2+ concentration in the absence of exogenous Mg2+. The presence of a Ca2+ pump on disk membranes is discussed in terms of its possible role in Ca2+ ion buffering during photoreceptor cell functioning.

  13. Arap1 Deficiency Causes Photoreceptor Degeneration in Mice

    Science.gov (United States)

    Moshiri, Ala; Humpal, Devin; Leonard, Brian C.; Imai, Denise M.; Tham, Addy; Bower, Lynette; Clary, Dave; Glaser, Thomas M.; Lloyd, K. C. Kent; Murphy, Christopher J.

    2017-01-01

    Purpose Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the control of GTPase-activating proteins (GAPs). Arap1 is an Arf-directed GAP that inhibits the trafficking of epidermal growth factor receptor (EGFR) to the early endosome, but the diversity of its functions is incompletely understood. The aim of this study was to determine the role of Arap1 in the mammalian retina. Methods Genetically engineered Arap1 knockout mice were screened for ocular abnormalities in the National Institutes of Health Knockout Mouse Production and Phenotyping (KOMP2) Project. Arap1 knockout and wild-type eyes were imaged using optical coherence tomography and fundus photography, and analyzed by immunohistochemistry. Results Arap1−/− mice develop a normal appearing retina, but undergo photoreceptor degeneration starting at 4 weeks postnatal age. The fundus appearance of mutants is notable for pigmentary changes, optic nerve pallor, vascular attenuation, and outer retinal thinning, reminiscent of retinitis pigmentosa in humans. Immunohistochemical studies suggest the cell death is predominantly in the outer nuclear layer. Functional evaluation of the retina by electroretinography reveals amplitudes are reduced. Arap1 is detected most notably in Müller glia, and not in photoreceptors, implicating a role for Müller glia in photoreceptor survival. Conclusions Arap1 is necessary for normal photoreceptor survival in mice, and may be a novel gene relevant to human retinal degenerative processes, although its mechanism is unknown. Further studies in this mouse model of retinal degeneration will give insights into the cellular functions and signaling pathways in which Arap1 participates. PMID:28324111

  14. Extracellular electrical activity from the photoreceptors of midge

    Indian Academy of Sciences (India)

    A A Babrekar; G R Kulkarni; B B Nath; P B Vidyasagar

    2004-09-01

    The ontogeny of photosensitivity has been studied in a holometabolous insect, the midge Chironomus ramosus. The life cycle of midges shifts from an aquatic environment to a non-aquatic environment. Extracellular electrical activity of photoreceptor organs was recorded at larval and adult stages. We found an increase in photosensitivity as the larva metamorphosed to the adult stage. This is the first report of changes in photosensitivity during the development of any insect described in an ecological context.

  15. Photoreceptor structure and function in patients with congenital achromatopsia.

    Science.gov (United States)

    Genead, Mohamed A; Fishman, Gerald A; Rha, Jungtae; Dubis, Adam M; Bonci, Daniela Maria O; Dubra, Alfredo; Stone, Edwin M; Neitz, Maureen; Carroll, Joseph

    2011-09-21

    To assess photoreceptor structure and function in patients with congenital achromatopsia. Twelve patients were enrolled. All patients underwent a complete ocular examination, spectral-domain optical coherence tomography (SD-OCT), full-field electroretinographic (ERG), and color vision testing. Macular microperimetry (MP; in four patients) and adaptive optics (AO) imaging (in nine patients) were also performed. Blood was drawn for screening of disease-causing genetic mutations. Mean (± SD) age was 30.8 (± 16.6) years. Mean best-corrected visual acuity was 0.85 (± 0.14) logarithm of the minimal angle of resolution (logMAR) units. Seven patients (58.3%) showed either an absent foveal reflex or nonspecific retinal pigment epithelium mottling to mild hypopigmentary changes on fundus examination. Two patients showed an atrophic-appearing macular lesion. On anomaloscopy, only 5 patients matched over the entire range from 0 to 73. SD-OCT examination showed a disruption or loss of the macular inner/outer segments (IS/OS) junction of the photoreceptors in 10 patients (83.3%). Seven of these patients showed an optically empty space at the level of the photoreceptors in the fovea. AO images of the photoreceptor mosaic were highly variable but significantly disrupted from normal. On ERG testing, 10 patients (83.3%) showed evidence of residual cone responses to a single-flash stimulus response. The macular MP testing showed that the overall mean retinal sensitivity was significantly lower than normal (12.0 vs. 16.9 dB, P achromatopsia should be useful in guiding selection of patients for future therapeutic trials as well as monitoring therapeutic response in these trials.

  16. Mitochondria Maintain Distinct Ca(2+) Pools in Cone Photoreceptors.

    Science.gov (United States)

    Giarmarco, Michelle M; Cleghorn, Whitney M; Sloat, Stephanie R; Hurley, James B; Brockerhoff, Susan E

    2017-02-22

    Ca(2+) ions have distinct roles in the outer segment, cell body, and synaptic terminal of photoreceptors. We tested the hypothesis that distinct Ca(2+) domains are maintained by Ca(2+) uptake into mitochondria. Serial block face scanning electron microscopy of zebrafish cones revealed that nearly 100 mitochondria cluster at the apical side of the inner segment, directly below the outer segment. The endoplasmic reticulum surrounds the basal and lateral surfaces of this cluster, but does not reach the apical surface or penetrate into the cluster. Using genetically encoded Ca(2+) sensors, we found that mitochondria take up Ca(2+) when it accumulates either in the cone cell body or outer segment. Blocking mitochondrial Ca(2+) uniporter activity compromises the ability of mitochondria to maintain distinct Ca(2+) domains. Together, our findings indicate that mitochondria can modulate subcellular functional specialization in photoreceptors.SIGNIFICANCE STATEMENT Ca(2+) homeostasis is essential for the survival and function of retinal photoreceptors. Separate pools of Ca(2+) regulate phototransduction in the outer segment, metabolism in the cell body, and neurotransmitter release at the synaptic terminal. We investigated the role of mitochondria in compartmentalization of Ca(2+) We found that mitochondria form a dense cluster that acts as a diffusion barrier between the outer segment and cell body. The cluster is surprisingly only partially surrounded by the endoplasmic reticulum, a key mediator of mitochondrial Ca(2+) uptake. Blocking the uptake of Ca(2+) by mitochondria causes redistribution of Ca(2+) throughout the cell. Our results show that mitochondrial Ca(2+) uptake in photoreceptors is complex and plays an essential role in normal function. Copyright © 2017 the authors 0270-6474/17/372061-12$15.00/0.

  17. A Novel In Vivo Model of Focal Light Emitting Diode-Induced Cone-Photoreceptor Phototoxicity: Neuroprotection Afforded by Brimonidine, BDNF, PEDF or bFGF

    Science.gov (United States)

    García-Ayuso, Diego; Alarcón-Martínez, Luis; Jiménez-López, Manuel; Bernal-Garro, José Manuel; Nieto-López, Leticia; Nadal-Nicolás, Francisco Manuel; Villegas-Pérez, María Paz; Wheeler, Larry A.; Vidal-Sanz, Manuel

    2014-01-01

    We have investigated the effects of light-emitting diode (LED)-induced phototoxicity (LIP) on cone-photoreceptors and their protection with brimonidine (BMD), brain-derived neurotrophic factor (BDNF), pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF). In anesthetized, dark adapted, adult albino rats a blue (400 nm) LED was placed perpendicular to the cornea (10 sec, 200 lux) and the effects were investigated using Spectral Domain Optical Coherence Tomography (SD-OCT) and/or analysing the retina in oriented cross-sections or wholemounts immune-labelled for L- and S-opsin and counterstained with the nuclear stain DAPI. The effects of topical BMD (1%) or, intravitreally injected BDNF (5 µg), PEDF (2 µg), CNTF (0.4 µg) or bFGF (1 µg) after LIP were examined on wholemounts at 7 days. SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4±84.5 µm (at 24 hours) to 1,407.7±52.8 µm (at 7 days). This region had a progressive thickness diminution from 183.4±5 µm (at 12 h) to 114.6±6 µm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118±842 or 661±125 for the LED exposed retinas (n = 7) and 14,040±1,860 or 2,255±193 for the fellow retinas (n = 7), respectively. BMD, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- or S-cones. We conclude that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model

  18. RNAi-mediated gene suppression in a GCAP1(L151F cone-rod dystrophy mouse model.

    Directory of Open Access Journals (Sweden)

    Li Jiang

    Full Text Available Dominant mutations occurring in the high-affinity Ca(2+-binding sites (EF-hands of the GUCA1A gene encoding guanylate cyclase-activating protein 1 (GCAP1 cause slowly progressing cone-rod dystrophy (CORD in a dozen families worldwide. We developed a nonallele-specific adeno-associated virus (AAV-based RNAi knockdown strategy to rescue the retina degeneration caused by GCAP1 mutations. We generated three genomic transgenic mouse lines expressing wildtype (WT and L151F mutant mouse GCAP1 with or without a C-terminal GFP fusion. Under control of endogenous regulatory elements, the transgenes were expressed specifically in mouse photoreceptors. GCAP1(L151F and GCAP1(L151F-GFP transgenic mice presented with a late onset and slowly progressive photoreceptor degeneration, similar to that observed in human GCAP1-CORD patients. Transgenic expression of WT GCAP1-EGFP in photoreceptors had no adverse effect. Toward therapy development, a highly effective anti-mGCAP1 shRNA, mG1hp4, was selected from four candidate shRNAs using an in-vitro screening assay. Subsequently a self-complementary (sc AAV serotype 2/8 expressing mG1hp4 was delivered subretinally to GCAP1(L151F-GFP transgenic mice. Knockdown of the GCAP1(L151F-GFP transgene product was visualized by fluorescence live imaging in the scAAV2/8-mG1hp4-treated retinas. Concomitant with the mutant GCAP1-GFP fusion protein, endogenous GCAP1 decreased as well in treated retinas. We propose nonallele-specific RNAi knockdown of GCAP1 as a general therapeutic strategy to rescue any GCAP1-based dominant cone-rod dystrophy in human patients.

  19. In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle.

    Science.gov (United States)

    Barsanti, Laura; Coltelli, Primo; Evangelista, Valtere; Passarelli, Vincenzo; Frassanito, Anna Maria; Vesentini, Nicoletta; Santoro, Fabrizio; Gualtieri, Paolo

    2009-01-01

    Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.

  20. Effect of Purified Murine NGF on Isolated Photoreceptors of a Rodent Developing Retinitis Pigmentosa

    Science.gov (United States)

    Rocco, Maria Luisa; Balzamino, Bijorn Omar; Petrocchi Passeri, Pamela; Micera, Alessandra; Aloe, Luigi

    2015-01-01

    A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies. PMID:25897972

  1. Usherin is required for maintenance of retinal photoreceptors and normal development of cochlear hair cells

    OpenAIRE

    2007-01-01

    Usher syndrome type IIA (USH2A), characterized by progressive photoreceptor degeneration and congenital moderate hearing loss, is the most common subtype of Usher syndrome. In this article, we show that the USH2A protein, also known as usherin, is an exceptionally large (≈600-kDa) matrix protein expressed specifically in retinal photoreceptors and developing cochlear hair cells. In mammalian photoreceptors, usherin is localized to a spatially restricted membrane microdomain at the apical inne...

  2. Viscoelasticity of suspensions of long, rigid rods

    NARCIS (Netherlands)

    Dhont, Jan K.G.; Briels, W.J.

    2003-01-01

    A microscopic theory for the viscoelastic behaviour of suspensions of rigid rods with excluded volume interactions is presented, which is valid in the asymptotic limit of very long and thin rods. Stresses arising from translational and rotational Brownian motion and direct interactions are calculate

  3. Study of the rod style SFRFQ structure

    CERN Document Server

    Yan Xue Qing; Chen J

    2002-01-01

    There is a problem about upper limit of energy in the RFQ structure, although it is a wonderful low-energy-suited high current accelerating structure. After proposing an improved rod style SFRFQ structure without reversed field, the author studies its energy gain and transverse motion. The rod style SFRFQ structure is roughly compared with diaphragm SFRFQ structure

  4. Three-dimensional structure of an invertebrate rhodopsin and basis for ordered alignment in the photoreceptor membrane.

    Science.gov (United States)

    Davies, A; Gowen, B E; Krebs, A M; Schertler, G F; Saibil, H R

    2001-11-30

    Invertebrate rhodopsins activate a G-protein signalling pathway in microvillar photoreceptors. In contrast to the transducin-cyclic GMP phosphodiesterase pathway found in vertebrate rods and cones, visual transduction in cephalopod (squid, octopus, cuttlefish) invertebrates is signalled via Gq and phospholipase C. Squid rhodopsin contains the conserved residues of the G-protein coupled receptor (GPCR) family, but has only 35% identity with mammalian rhodopsins. Unlike vertebrate rhodopsins, cephalopod rhodopsin is arranged in an ordered lattice in the photoreceptor membranes. This organization confers sensitivity to the plane of polarized light and also provides the optimal orientation of the linear retinal chromophores in the cylindrical microvillar membranes for light capture. Two-dimensional crystals of squid rhodopsin show a rectilinear arrangement that is likely to be related to the alignment of rhodopsins in vivo.Here, we present a three-dimensional structure of squid rhodopsin determined by cryo-electron microscopy of two-dimensional crystals. Docking the atomic structure of bovine rhodopsin into the squid density map shows that the helix packing and extracellular plug structure are conserved. In addition, there are two novel structural features revealed by our map. The linear lattice contact appears to be made by the transverse C-terminal helix lying on the cytoplasmic surface of the membrane. Also at the cytoplasmic surface, additional density may correspond to a helix 5-6 loop insertion found in most GPCRs relative to vertebrate rhodopsins. The similarity supports the conservation in structure of rhodopsins (and other G-protein-coupled receptors) from phylogenetically distant organisms. The map provides the first indication of the structural basis for rhodopsin alignment in the microvillar membrane.

  5. Phase behavior and structure formation of hairy-rod supramolecules

    NARCIS (Netherlands)

    Subbotin, A; Stepanyan, R; Knaapila, M; Ikkala, O; ten Brinke, G

    2003-01-01

    Phase behavior and microstructure formation of rod and coil molecules, which can associate to form hairy-rod polymeric supramolecules, are addressed theoretically. Association induces considerable compatibility enhancement between the rod and coil molecules and various microscopically ordered struct

  6. Biosynthesis of glycerolipid molecular species in photoreceptor membranes of frog retina

    Energy Technology Data Exchange (ETDEWEB)

    Wiegand, R.D.; Louie, K.; Anderson, R.E. (Baylor College of Medicine, Houston, TX (USA))

    1987-05-01

    Phospholipid (PL) molecular species of vertebrate retinal photoreceptor cells are unique in that they contain two polyunsaturated fatty acids per molecule. Docosahexaenoic acid (22:6 {omega}3) is the major component of these dipolyunsaturate species (DPS), which also contain 20:4{omega}6, 22:4{omega}6, 22:5{omega}6, and 22:5{omega}3. We have studied the de novo synthesis and metabolism of the (DPS) and other PL molecular species in frog rod outer segments (ROS) following intravitreal injection of 2-({sup 3}H)-glycerol. At 1, 2, 4, and 8 days after injection, ROS were prepared, PL extracted, and phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) isolated. PC, PE, and PS were converted to diglycerides (DG's) with phospholipase C. DG's were derivatized, fractionated into molecular species by HPLC, quantitated, and counted for radioactivity. The following were observed: (1) Specific activities (SA) of the PC DPS were 3-5 times higher than the same species in either PE or PS. (2) SA of the PC monopolyunsaturate species (MPS) (species which contain 22:6{omega}3 and/or 16:0 or 18:0) were 3-5 times lower than the SA of the PC DPS. In contrast, SA of PE MPS were 2-5 times higher than the SA of the PE DPS. (3) The major PS MPS synthesized contained 18:0 and 22:6{omega}3. SA of that species were similar to SA of the PS DPS. The data support the suggestion that PC DPS are synthesized and/or incorporated in ROS at a greater rate than the same species in either PE or PS. Our study thus provides evidence for different rates of synthesis and/or incorporation of the various molecular species of PC, PE, and PS in ROS.

  7. Cobalamin C Deficiency Shows a Rapidly Progressing Maculopathy With Severe Photoreceptor and Ganglion Cell Loss.

    Science.gov (United States)

    Bonafede, Lucas; Ficicioglu, Can H; Serrano, Leona; Han, Grace; Morgan, Jessica I W; Mills, Monte D; Forbes, Brian J; Davidson, Stefanie L; Binenbaum, Gil; Kaplan, Paige B; Nichols, Charles W; Verloo, Patrick; Leroy, Bart P; Maguire, Albert M; Aleman, Tomas S

    2015-12-01

    To describe in detail the retinal structure and function of a group of patients with cobalamin C (cblC) disease. Patients (n = 11, age 4 months to 15 years) with cblC disease (9/11, early onset) diagnosed by newborn screening underwent complete ophthalmic examinations, fundus photography, near-infrared reflectance imaging, and spectral-domain optical coherence tomography (SD-OCT). Electroretinograms (ERGs) were performed in a subset of patients. Patients carried homozygous or compound heterozygote mutations in the methylmalonic aciduria and homocystinuria type C (MMACHC) gene. Late-onset patients had a normal exam. All early-onset patients showed a maculopathy; older subjects had a retina-wide degeneration (n = 4; >7 years of age). In general, retinal changes were first observed before 1 year of age and progressed within months to a well-established maculopathy. Pseudocolobomas were documented in three patients. Measurable visual acuities ranged from 20/200 to 20/540. Nystagmus was present in 8/11 patients; 5/6 patients had normal ERGs; 1/6 had reduced rod-mediated responses. Spectral-domain OCT showed macular thinning, with severe ganglion cell layer (GCL) and outer nuclear layer (ONL) loss. Inner retinal thickening was observed in areas of total GCL/ONL loss. A normal lamination pattern in the peripapillary nasal retina was often seen despite severe central and/or retina-wide disease. Patients with early-onset cblC and MMACHC mutations showed an early-onset, unusually fast-progressing maculopathy with severe central ONL and GCL loss. An abnormally thickened inner retina supports a remodeling response to both photoreceptor and ganglion cell degeneration and/or an interference with normal development in early-onset cblC.

  8. Eulerian Formulation of Spatially Constrained Elastic Rods

    Science.gov (United States)

    Huynen, Alexandre

    Slender elastic rods are ubiquitous in nature and technology. For a vast majority of applications, the rod deflection is restricted by an external constraint and a significant part of the elastic body is in contact with a stiff constraining surface. The research work presented in this doctoral dissertation formulates a computational model for the solution of elastic rods constrained inside or around frictionless tube-like surfaces. The segmentation strategy adopted to cope with this complex class of problems consists in sequencing the global problem into, comparatively simpler, elementary problems either in continuous contact with the constraint or contact-free between their extremities. Within the conventional Lagrangian formulation of elastic rods, this approach is however associated with two major drawbacks. First, the boundary conditions specifying the locations of the rod centerline at both extremities of each elementary problem lead to the establishment of isoperimetric constraints, i.e., integral constraints on the unknown length of the rod. Second, the assessment of the unilateral contact condition requires, in principle, the comparison of two curves parametrized by distinct curvilinear coordinates, viz. the rod centerline and the constraint axis. Both conspire to burden the computations associated with the method. To streamline the solution along the elementary problems and rationalize the assessment of the unilateral contact condition, the rod governing equations are reformulated within the Eulerian framework of the constraint. The methodical exploration of both types of elementary problems leads to specific formulations of the rod governing equations that stress the profound connection between the mechanics of the rod and the geometry of the constraint surface. The proposed Eulerian reformulation, which restates the rod local equilibrium in terms of the curvilinear coordinate associated with the constraint axis, describes the rod deformed configuration

  9. Beneficial effects of combined benazepril-amlodipine on cardiac nitric oxide, cGMP, and TNF-alpha production after cardiac ischemia.

    Science.gov (United States)

    Siragy, Helmy M; Xue, Chun; Webb, Randy L

    2006-05-01

    The aim of this study was to determine if myocardial inflammation is increased after myocardial ischemia and whether angiotensin-converting enzyme inhibitors, calcium channel blockers, or diuretics decrease mediators of inflammation in rats with induced myocardial ischemia. Changes in cardiac interstitial fluid (CIF) levels of nitric oxide metabolites (NOX), cyclic guanosine 3',5'-monophosphate (cGMP), angiotensin II (Ang II), and tumor necrosis factor-alpha (TNF-alpha) were monitored with/without oral administration of benazepril, amlodipine, combined benazepril-amlodipine, or hydrochlorothiazide. Using a microdialysis technique, levels of several mediators of inflammation were measured after sham operation or 30-minute occlusion of the left anterior descending coronary artery. Compared with sham animals, levels of CIF NOX and cGMP were decreased in animals with ischemia (P Benazepril or amlodipine significantly increased NOX levels (P benazepril significantly increased cGMP (P benazepril-amlodipine further increased CIF NOX and cGMP (P Amlodipine alone, benazepril alone, or combined benazepril-amlodipine significantly reduced TNF-alpha (P benazepril-amlodipine may be beneficial for managing cardiac ischemia.

  10.  Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism

    Directory of Open Access Journals (Sweden)

    Edyta Makuch

    2012-07-01

    Full Text Available  PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme’s intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.

  11. cAMP and cGMP in nasal mucus related to severity of smell loss in patients with smell dysfunction.

    Science.gov (United States)

    Henkin, R I; Velicu, I

    2008-01-01

    To evaluate nasal mucus levels of cAMP and cGMP in patients with taste and smell dysfunction with respect to severity of their smell loss. cAMP and cGMP were measured in nasal mucus using a sensitive spectrophotometric 96 plate ELISA technique. Smell loss was measured in patients with taste and smell dysfunction by standardized psychophysical measurements of olfactory function and classified by severity of loss into four types from most severe to least severe such that anosmia > Type I hyposmia > Type II hyposmia > Type III hyposmia. Measurements of nasal mucus cyclic nucleotides and smell loss were made independently. As smell loss severity increased stepwise cAMP and cGMP levels decreased stepwise [cAMP, cGMP (in pmol/ml); anosmia - 0.004, 0.008: Type I hyposmia - 0.12+/-0.03, 0.10+/-0.03: Type II hyposmia - 0.15+/-0.02, 0.16+/-0.01: Type III hyposmia - 0.23+/-0.05, 0.20+/-0.15]. These results confirm the association of biochemical changes in cyclic nucleotides with systematic losses of smell acuity. These results confirm the usefulness of the psychophysical methods we defined to determine the systematic classification of smell loss severity. These changes can form the basis for the biochemical definition of smell loss among some patients with smell loss as well as for their therapy.

  12. Effects of Kaempferia parviflora Wall. Ex. Baker and sildenafil citrate on cGMP level, cardiac function, and intracellular Ca2+ regulation in rat hearts.

    Science.gov (United States)

    Weerateerangkul, Punate; Palee, Siripong; Chinda, Kroekkiat; Chattipakorn, Siriporn C; Chattipakorn, Nipon

    2012-09-01

    Although Kaempferia parviflora extract (KPE) and its flavonoids have positive effects on the nitric oxide (NO) signaling pathway, its mechanisms on the heart are still unclear. Because our previous studies demonstrated that KPE decreased defibrillation efficacy in swine similar to that of sildenafil citrate, the phosphodiesterase-5 inhibitor, it is possible that KPE may affect the cardiac NO signaling pathway. In the present study, the effects of KPE and sildenafil citrate on cyclic guanosine monophosphate (cGMP) level, modulation of cardiac function, and Ca transients in ventricular myocytes were investigated. In a rat model, cardiac cGMP level, cardiac function, and Ca transients were measured before and after treatment with KPE and sildenafil citrate. KPE significantly increased the cGMP level and decreased cardiac function and Ca transient. These effects were similar to those found in the sildenafil citrate-treated group. Furthermore, the nonspecific NOS inhibitor could abolish the effects of KPE and sildenafil citrate on Ca transient. KPE has positive effect on NO signaling in the heart, resulting in an increased cGMP level, similar to that of sildenafil citrate. This effect was found to influence the physiology of normal heart via the attenuation of cardiac function and the reduction of Ca transient in ventricular myocytes.

  13. Role of kinesin heavy chain in Crumbs localization along the rhabdomere elongation in Drosophila photoreceptor.

    Directory of Open Access Journals (Sweden)

    Garrett P League

    Full Text Available BACKGROUND: Crumbs (Crb, a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ, and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc, a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors. METHODOLOGY/PRINCIPAL FINDINGS: Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc's gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors. CONCLUSIONS/SIGNIFICANCE: In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in

  14. Morphoelastic rods Part II: Growing birods

    Science.gov (United States)

    Lessinnes, Thomas; Moulton, Derek E.; Goriely, Alain

    2017-03-01

    The general problem of determining the shape and response of two attached growing elastic Kirchhoff rods is considered. A description of the kinematics of the individual interacting rods is introduced. Each rod has a given intrinsic shape and constitutive laws, and a map associating points on the two rods is defined. The resulting filamentary structure, a growing birod, can be seen as a new filamentary structure. This kinematic description is used to derive the general equilibrium equations for the shape of the rods under loads, or equivalently, for the new birod. It is shown that, in general, the birod is not simply a Kirchhoff rod but rather, due to the internal constraints, new effects can appear. The two-dimensional restriction is then considered explicitly and the limit for small deformation is shown to be equivalent to the classic Timsohenko bi-metallic strip problem. A number of examples and applications are presented. In particular, the problem of two attached rods with intrinsic helical shape and uniform growth is computed in detail and a host of new interesting solutions and bifurcations are observed.

  15. Granular materials interacting with thin flexible rods

    Science.gov (United States)

    Neto, Alfredo Gay; Campello, Eduardo M. B.

    2017-04-01

    In this work, we develop a computational model for the simulation of problems wherein granular materials interact with thin flexible rods. We treat granular materials as a collection of spherical particles following a discrete element method (DEM) approach, while flexible rods are described by a large deformation finite element (FEM) rod formulation. Grain-to-grain, grain-to-rod, and rod-to-rod contacts are fully permitted and resolved. A simple and efficient strategy is proposed for coupling the motion of the two types (discrete and continuum) of materials within an iterative time-stepping solution scheme. Implementation details are shown and discussed. Validity and applicability of the model are assessed by means of a few numerical examples. We believe that robust, efficiently coupled DEM-FEM schemes can be a useful tool to the simulation of problems wherein granular materials interact with thin flexible rods, such as (but not limited to) bombardment of grains on beam structures, flow of granular materials over surfaces covered by threads of hair in many biological processes, flow of grains through filters and strainers in various industrial segregation processes, and many others.

  16. Magnetically controlled growing rods for scoliosis surgery.

    Science.gov (United States)

    Metkar, Umesh; Kurra, Swamy; Quinzi, David; Albanese, Stephen; Lavelle, William F

    2017-02-01

    Early onset scoliosis can be both a disfiguring as well as a life threatening condition. When more conservative treatments fail, pediatric spinal surgeons are forced to consider operative interventions. Traditionally, these interventions have involved the insertion of a variety of implants into the patient with a limited number of anchor points controlling the spine. In the past, these pediatric patients have had multiple surgeries for elective lengthening of these devices to facilitate their growth while attempting to control the scoliosis. These patients often experience a physical and emotional toll from their multiple repeated surgeries. Growing spine techniques have also had a noted high complication rate due to implant dislodgement and infections. Recently, the development of non-invasively, self-lengthening growing rods has occurred. These devices have the potential to allow for the devices to be lengthened magnetically in a conscious patient in the surgeon's office. Areas covered: This review summarized previously published articles in the English literature using a key word search in PubMed for: 'magnetically controlled growing rods', 'Magec rods', 'magnetic growing rods' and 'growing rods'. Expert commentary: Magnetically controlled growing rods have an advantage over growing rods in lengthening the growing spine in the absence of repetitive surgeries.

  17. Control Rod Malfunction at the NRAD Reactor

    Energy Technology Data Exchange (ETDEWEB)

    Thomas L. Maddock

    2010-05-01

    The neutron Radiography Reactor (NRAD) is a training, research, and isotope (TRIGA) reactor located at the INL. The reactor is normally shut down by the insertion of three control rods that drop into the core when power is removed from electromagnets. During a routine shutdown, indicator lights on the console showed that one of the control rods was not inserted. It was initially thought that the indicator lights were in error because of a limit switch that was out of adjustment. Through further testing, it was determined that the control rod did not drop when the scram switch was initially pressed. The control rod anomaly led to a six month shutdown of the reactor and an in depth investigation of the reactor protective system. The investigation looked into: scram switch operation, console modifications, and control rod drive mechanisms. A number of latent issues were discovered and corrected during the investigation. The cause of the control rod malfunction was found to be a buildup of corrosion in the control rod drive mechanism. The investigation resulted in modifications to equipment, changes to both operation and maintenance procedures, and additional training. No reoccurrences of the problem have been observed since corrective actions were implemented.

  18. Estimation of irradiated control rod worth

    Energy Technology Data Exchange (ETDEWEB)

    Varvayanni, M., E-mail: melina@ipta.demokritos.g [NCSR ' DEMOKRITOS' , PO Box 60228, 15310 Aghia Paraskevi (Greece); Catsaros, N. [NCSR ' DEMOKRITOS' , PO Box 60228, 15310 Aghia Paraskevi (Greece); Antonopoulos-Domis, M. [School of Electrical and Computer Engineering, Aristotle University of Thessaloniki, Thessaloniki (Greece)

    2009-11-15

    When depleted control rods are planned to be used in new core configurations, their worth has to be accurately predicted in order to deduce key design and safety parameters such as the available shutdown margin. In this work a methodology is suggested for the derivation of the distributed absorbing capacity of a depleted rod, useful in the case that the level of detail that is known about the irradiation history of the control rod does not allow an accurate calculation of the absorber's burnup. The suggested methodology is based on measurements of the rod's worth carried out in the former core configuration and on corresponding calculations based on the original (before first irradiation) absorber concentration. The methodology is formulated for the general case of the multi-group theory; it is successfully tested for the one-group approximation, for a depleted control rod of the Greek Research Reactor, containing five neutron absorbers. The computations reproduce satisfactorily the irradiated rod worth measurements, practically eliminating the discrepancy of the total rod worth, compared to the computations based on the nominal absorber densities.

  19. Granular materials interacting with thin flexible rods

    Science.gov (United States)

    Neto, Alfredo Gay; Campello, Eduardo M. B.

    2016-01-01

    In this work, we develop a computational model for the simulation of problems wherein granular materials interact with thin flexible rods. We treat granular materials as a collection of spherical particles following a discrete element method (DEM) approach, while flexible rods are described by a large deformation finite element (FEM) rod formulation. Grain-to-grain, grain-to-rod, and rod-to-rod contacts are fully permitted and resolved. A simple and efficient strategy is proposed for coupling the motion of the two types (discrete and continuum) of materials within an iterative time-stepping solution scheme. Implementation details are shown and discussed. Validity and applicability of the model are assessed by means of a few numerical examples. We believe that robust, efficiently coupled DEM-FEM schemes can be a useful tool to the simulation of problems wherein granular materials interact with thin flexible rods, such as (but not limited to) bombardment of grains on beam structures, flow of granular materials over surfaces covered by threads of hair in many biological processes, flow of grains through filters and strainers in various industrial segregation processes, and many others.

  20. Calcium-dependent potassium current in barnacle photoreceptor

    OpenAIRE

    1981-01-01

    When barnacle lateral eye photoreceptors are depolarized to membrane potentials of 0 to +50 mV in the dark, the plot of outward current through the cell membrane against time has two distinct maxima. The first maximum occurs 5-10 ms after the depolarization began. The current then decays to a minimum at approximately 500 ms after the onset of depolarization, and then increases to a second maximum 4-6 s after the depolarization began. If depolarization is maintained, the current again decays t...

  1. Constitutively active UVR8 photoreceptor variant in Arabidopsis

    OpenAIRE

    2013-01-01

    Sunlight is an essential environmental factor for photosynthetic plants and ultimately for life on Earth, which is sustained through plants as fundamental source of food. However, plants have a love/hate relationship with sunlight and must be protected from potentially harmful UV-B radiation. The UV-B photoreceptor UVR8 is of great importance in mounting UV-protective responses and thus for survival in sunlight. Based on our understanding of UVR8 signaling, we have engineered a UVR8 variant t...

  2. Tipping time of a quantum rod

    Energy Technology Data Exchange (ETDEWEB)

    Parrikar, Onkar [Birla Institute of Technology and Science-Pilani, Goa campus, Zuarinagar, Goa 4032726 (India)], E-mail: onkarsp@gmail.com

    2010-03-15

    The behaviour of a quantum rod, pivoted at its lower end on an impenetrable floor and restricted to moving in the vertical plane under the gravitational potential, is studied analytically under the approximation that the rod is initially localized to a 'small-enough' neighbourhood around the point of classical unstable equilibrium. It is shown that the rod evolves out of this neighbourhood. The time required for this to happen, i.e. the tipping time, is calculated using the semi-classical path integral. It is shown that equilibrium is recovered in the classical limit, and that our calculations are consistent with the uncertainty principle.

  3. High temperature control rod assembly

    Energy Technology Data Exchange (ETDEWEB)

    Vollman, R.E.

    1991-12-24

    This patent describes a control rod assembly for use in nuclear reactor control. It comprises segments, each the segment being made of a graphite composite material, each the segment having a chamber for containing neutron-absorbing material, wherein the chamber compromises a hollow cylindrical sleeve having a first end formed with an opening for receiving the neutron-absorbing material, and having a second end formed with a sleeve bore and an outer sleeve surface; a cylindrical weight-bearing support post positioned substantially centrally of the sleeve, the support post having a first end formed as a ball surface portion and a second end formed as a ball surface portion and a second end formed as a shaft, the shaft being engageable with the sleeve bore for rigidly coupling the support post axially within the hollow sleeve, a hollow cylindrical collar having a socket lip portion correspondingly shaped to receive the ball surface portion of an adjacent support post, and having an inner surface for engaging the outer sleeve surface on the second end of the sleeve to rigidly couple the collar to the sleeve.

  4. Ionic currents underlying difference in light response between type A and type B photoreceptors.

    Science.gov (United States)

    Blackwell, K T

    2006-05-01

    In Hermissenda crassicornis, the memory of light associated with turbulence is stored as changes in intrinsic and synaptic currents in both type A and type B photoreceptors. These photoreceptor types exhibit qualitatively different responses to light and current injection, and these differences shape the spatiotemporal firing patterns that control behavior. Thus the objective of the study was to identify the mechanisms underlying these differences. The approach was to develop a type B model that reproduced characteristics of type B photoreceptors recorded in vitro, and then to create a type A model by modifying a select number of ionic currents. Comparison of type A models with characteristics of type A photoreceptors recorded in vitro revealed that type A and type B photoreceptors have five main differences, three that have been characterized experimentally and two that constitute hypotheses to be tested with experiments in the future. The three differences between type A and type B photoreceptors previously characterized include the inward rectifier current, the fast sodium current, and conductance of calcium-dependent and transient potassium channels. Two additional changes were required to produce a type A photoreceptor model. The very fast firing frequency observed during the first second after light onset required a faster time constant of activation of the delayed rectifier. The fast spike adaptation required a fast, noninactivating calcium-dependent potassium current. Because these differences between type A and type B photoreceptors have not been confirmed in comparative experiments, they constitute hypotheses to be tested with future experiments.

  5. Large variation among photoreceptors as the basis of visual flexibility in the common backswimmer

    Science.gov (United States)

    Immonen, Esa-Ville; Ignatova, Irina; Gislen, Anna; Warrant, Eric; Vähäsöyrinki, Mikko; Weckström, Matti; Frolov, Roman

    2014-01-01

    The common backswimmer, Notonecta glauca, uses vision by day and night for functions such as underwater prey animal capture and flight in search of new habitats. Although previous studies have identified some of the physiological mechanisms facilitating such flexibility in the animal's vision, neither the biophysics of Notonecta photoreceptors nor possible cellular adaptations are known. Here, we studied Notonecta photoreceptors using patch-clamp and intracellular recording methods. Photoreceptor size (approximated by capacitance) was positively correlated with absolute sensitivity and acceptance angles. Information rate measurements indicated that large and more sensitive photoreceptors performed better than small ones. Our results suggest that backswimmers are adapted for vision in both dim and well-illuminated environments by having open-rhabdom eyes with large intrinsic variation in absolute sensitivity among photoreceptors, exceeding those found in purely diurnal or nocturnal species. Both electrophysiology and microscopic analysis of retinal structure suggest two retinal subsystems: the largest peripheral photoreceptors provide vision in dim light and the smaller peripheral and central photoreceptors function primarily in sunlight, with light-dependent pigment screening further contributing to adaptation in this system by dynamically recruiting photoreceptors with varying sensitivity into the operational pool. PMID:25274359

  6. Evidence for dynamic network regulation of Drosophila photoreceptor function from mutants lacking the neurotransmitter histamine

    Directory of Open Access Journals (Sweden)

    An eDau

    2016-03-01

    Full Text Available Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. Here, we investigate this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the hdcJK910 mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that hdcJK910 photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected hdcJK910 photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to hdcJK910 R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in hdcJK910 mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons.

  7. Evidence for Dynamic Network Regulation of Drosophila Photoreceptor Function from Mutants Lacking the Neurotransmitter Histamine.

    Science.gov (United States)

    Dau, An; Friederich, Uwe; Dongre, Sidhartha; Li, Xiaofeng; Bollepalli, Murali K; Hardie, Roger C; Juusola, Mikko

    2016-01-01

    Synaptic feedback from interneurons to photoreceptors can help to optimize visual information flow by balancing its allocation on retinal pathways under changing light conditions. But little is known about how this critical network operation is regulated dynamically. Here, we investigate this question by comparing signaling properties and performance of wild-type Drosophila R1-R6 photoreceptors to those of the hdc (JK910) mutant, which lacks the neurotransmitter histamine and therefore cannot transmit information to interneurons. Recordings show that hdc (JK910) photoreceptors sample similar amounts of information from naturalistic stimulation to wild-type photoreceptors, but this information is packaged in smaller responses, especially under bright illumination. Analyses reveal how these altered dynamics primarily resulted from network overload that affected hdc (JK910) photoreceptors in two ways. First, the missing inhibitory histamine input to interneurons almost certainly depolarized them irrevocably, which in turn increased their excitatory feedback to hdc (JK910) R1-R6s. This tonic excitation depolarized the photoreceptors to artificially high potentials, reducing their operational range. Second, rescuing histamine input to interneurons in hdc (JK910) mutant also restored their normal phasic feedback modulation to R1-R6s, causing photoreceptor output to accentuate dynamic intensity differences at bright illumination, similar to the wild-type. These results provide mechanistic explanations of how synaptic feedback connections optimize information packaging in photoreceptor output and novel insight into the operation and design of dynamic network regulation of sensory neurons.

  8. Electrical coupling of neuro-ommatidial photoreceptor cells in the blowfly

    NARCIS (Netherlands)

    Hateren, J.H. van

    1986-01-01

    A new method of microstimulation of the blowfly eye using corneal neutralization was applied to the 6 peripheral photoreceptor cells (R1-R6) connected to one neuro-ommatidium (and thus looking into the same direction), whilst the receptor potential of a dark-adapted photoreceptor cell was recorded b

  9. Cellular elements for seeing in the dark: voltage-dependent conductances in cockroach photoreceptors

    Directory of Open Access Journals (Sweden)

    Salmela Iikka

    2012-08-01

    Full Text Available Abstract Background The importance of voltage-dependent conductances in sensory information processing is well-established in insect photoreceptors. Here we present the characterization of electrical properties in photoreceptors of the cockroach (Periplaneta americana, a nocturnal insect with a visual system adapted for dim light. Results Whole-cell patch-clamped photoreceptors had high capacitances and input resistances, indicating large photosensitive rhabdomeres suitable for efficient photon capture and amplification of small photocurrents at low light levels. Two voltage-dependent potassium conductances were found in the photoreceptors: a delayed rectifier type (KDR and a fast transient inactivating type (KA. Activation of KDR occurred during physiological voltage responses induced by light stimulation, whereas KA was nearly fully inactivated already at the dark resting potential. In addition, hyperpolarization of photoreceptors activated a small-amplitude inward-rectifying (IR current mediated at least partially by chloride. Computer simulations showed that KDR shapes light responses by opposing the light-induced depolarization and speeding up the membrane time constant, whereas KA and IR have a negligible role in the majority of cells. However, larger KA conductances were found in smaller and rapidly adapting photoreceptors, where KA could have a functional role. Conclusions The relative expression of KA and KDR in cockroach photoreceptors was opposite to the previously hypothesized framework for dark-active insects, necessitating further comparative work on the conductances. In general, the varying deployment of stereotypical K+ conductances in insect photoreceptors highlights their functional flexibility in neural coding.

  10. On the Effective Optical Density of the Pupil Mechanism in Fly Photoreceptors

    NARCIS (Netherlands)

    Roebroek, Jos G.H.; Stavenga, Doekele G.

    1990-01-01

    A simple electrophysiological method is described for determining the effective optical density of the intracellular pupil mechanism of insect photoreceptor ceils. The method depends on the fact that the photoreceptors can not only be illuminated in the normal, orthodromic way, but also antidromical

  11. Photoreceptor processing speed and input resistance changes during light adaptation correlate with spectral class in the bumblebee, Bombus impatiens.

    Directory of Open Access Journals (Sweden)

    Peter Skorupski

    Full Text Available Colour vision depends on comparison of signals from photoreceptors with different spectral sensitivities. However, response properties of photoreceptor cells may differ in ways other than spectral tuning. In insects, for example, broadband photoreceptors, with a major sensitivity peak in the green region of the spectrum (>500 nm, drive fast visual processes, which are largely blind to chromatic signals from more narrowly-tuned photoreceptors with peak sensitivities in the blue and UV regions of the spectrum. In addition, electrophysiological properties of the photoreceptor membrane may result in differences in response dynamics of photoreceptors of similar spectral class between species, and different spectral classes within a species. We used intracellular electrophysiological techniques to investigate response dynamics of the three spectral classes of photoreceptor underlying trichromatic colour vision in the bumblebee, Bombus impatiens, and we compare these with previously published data from a related species, Bombus terrestris. In both species, we found significantly faster responses in green, compared with blue- or UV-sensitive photoreceptors, although all 3 photoreceptor types are slower in B. impatiens than in B. terrestris. Integration times for light-adapted B. impatiens photoreceptors (estimated from impulse response half-width were 11.3 ± 1.6 ms for green photoreceptors compared with 18.6 ± 4.4 ms and 15.6 ± 4.4 for blue and UV, respectively. We also measured photoreceptor input resistance in dark- and light-adapted conditions. All photoreceptors showed a decrease in input resistance during light adaptation, but this decrease was considerably larger (declining to about 22% of the dark value in green photoreceptors, compared to blue and UV (41% and 49%, respectively. Our results suggest that the conductances associated with light adaptation are largest in green photoreceptors, contributing to their greater temporal processing speed

  12. Impact of AD995 alumina rods

    Energy Technology Data Exchange (ETDEWEB)

    Chhabildas, L.C.; Furnish, M.D.; Reinhart, W.D. [Sandia National Labs., Albuquerque, NM (United States); Grady, D.E. [Applied Research Associates, Inc., Albuquerque, NM (United States)

    1997-10-01

    Gas guns and velocity interferometric techniques have been used to determine the loading behavior of an AD995 alumina rod 19 mm in diameter by 75 mm and 150 mm long, respectively. Graded-density materials were used to impact both bare and sleeved alumina rods while the velocity interferometer was used to monitor the axial-velocity of the free end of the rods. Results of these experiments demonstrate that (1) a time-dependent stress pulse generated during impact allows an efficient transition from the initial uniaxial strain loading to a uniaxial stress state as the stress pulse propagates through the rod, and (2) the intermediate loading rates obtained in this configuration lie between split Hopkinson bar and shock-loading techniques.

  13. Computer simulation of rod-sphere mixtures

    CERN Document Server

    Antypov, D

    2003-01-01

    Results are presented from a series of simulations undertaken to investigate the effect of adding small spherical particles to a fluid of rods which would otherwise represent a liquid crystalline (LC) substance. Firstly, a bulk mixture of Hard Gaussian Overlap particles with an aspect ratio of 3:1 and hard spheres with diameters equal to the breadth of the rods is simulated at various sphere concentrations. Both mixing-demixing and isotropic-nematic transition are studied using Monte Carlo techniques. Secondly, the effect of adding Lennard-Jones particles to an LC system modelled using the well established Gay-Berne potential is investigated. These rod-sphere mixtures are simulated using both the original set of interaction parameters and a modified version of the rod-sphere potential proposed in this work. The subject of interest is the internal structure of the binary mixture and its dependence on density, temperature, concentration and various parameters characterising the intermolecular interactions. Both...

  14. Bouncing Balls and Hot Rod Races.

    Science.gov (United States)

    Tibbs, Peggy; Sherrill, Donna

    This paper presents the Bouncing Ball Experiment which models quadratic and exponential functions, and the Hot Rod Races activity that explores velocity and acceleration. Activities include directions for the use of TI-82 and TI-83 calculators. (YDS)

  15. Response Function of the Crayfish Caudal Photoreceptor to Hydrodynamic Stimuli

    Science.gov (United States)

    Breite, Sally; Bahar, Sonya; Neiman, Alexander; Moss, Frank

    2002-03-01

    In its abdominal 6th ganglion the crayfish houses 2 light-sensitive neurons (caudal photoreceptors, or CPRs). It is known that these neurons work in tandem with a mechanosensory system of tiny hairs spread across the tailfan, which make synaptic contact with the photoreceptors. A stochastic resonance effect has been shown in this system in which light enhances the transduction of a weak, periodic mechanosensory (hydrodynamic) stimulus. It is not known, however, whether an optimal response from the CPR is induced by a single sine wave cycle or some other waveform. We have experimentally investigated this favorable waveform by driving a tailfan preparation with mechanical 10 Hz correlated Ornstein-Uhlenbeck noise and calculating the response function from the spike-triggered average of the applied noise waveform. We will discuss differences in the shape of the optimal waveform under dark and light conditions, as well as what seems to be a noticeable difference in the magnitude of the animals' response to a noisy stimulus in comparison with a periodic stimulus.

  16. Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.

    Science.gov (United States)

    Bulgakova, Natalia A; Rentsch, Michaela; Knust, Elisabeth

    2010-11-15

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

  17. Double-clad nuclear fuel safety rod

    Science.gov (United States)

    McCarthy, William H.; Atcheson, Donald B.; Vaidyanathan, Swaminathan

    1984-01-01

    A device for shutting down a nuclear reactor during an undercooling or overpower event, whether or not the reactor's scram system operates properly. This is accomplished by double-clad fuel safety rods positioned at various locations throughout the reactor core, wherein melting of a secondary internal cladding of the rod allows the fuel column therein to shift from the reactor core to place the reactor in a subcritical condition.

  18. Microelectrophoresis of Silica Rods Using Confocal Microscopy.

    Science.gov (United States)

    Bakker, Henriëtte E; Besseling, Thijs H; Wijnhoven, Judith E G J; Helfferich, Peter H; van Blaaderen, Alfons; Imhof, Arnout

    2017-01-31

    The electrophoretic mobility and the zeta potential (ζ) of fluorescently labeled colloidal silica rods, with an aspect ratio of 3.8 and 6.1, were determined with microelectrophoresis measurements using confocal microscopy. In the case where the colloidal particles all move at the same speed parallel to the direction of the electric field, we record a xyz-stack over the whole depth of the capillary. This method is faster and more robust compared to taking xyt-series at different depths inside the capillary to obtain the parabolic flow profile, as was done in previous work from our group. In some cases, rodlike particles do not move all at the same speed in the electric field, but exhibit a velocity that depends on the angle between the long axis of the rod and the electric field. We measured the orientation-dependent velocity of individual silica rods during electrophoresis as a function of κa, where κ(-1) is the double layer thickness and a is the radius of the rod associated with the diameter. Thus, we determined the anisotropic electrophoretic mobility of the silica rods with different sized double layers. The size of the double layer was tuned by suspending silica rods in different solvents at different electrolyte concentrations. We compared these results with theoretical predictions. We show that even at already relatively high κa when the Smoluchowski limiting law is assumed to be valid (κa > 10), an orientation dependent velocity was measured. Furthermore, we observed that at decreasing values of κa the anisotropy in the electrophoretic mobility of the rods increases. However, in low polar solvents with κa < 1, this trend was reversed: the anisotropy in the electrophoretic mobility of the rods decreased. We argue that this decrease is due to end effects, which was already predicted theoretically. When end effects are not taken into account, this will lead to strong underestimation of the experimentally determined zeta potential.

  19. High Power Performance of Rod Fiber Amplifiers

    DEFF Research Database (Denmark)

    Johansen, Mette Marie; Michieletto, Mattia; Kristensen, Torben

    2015-01-01

    An improved version of the DMF rod fiber is tested in a high power setup delivering 360W of stable signal power. Multiple testing degrades the fiber and transverse modal instability threshold from >360W to ~290W.......An improved version of the DMF rod fiber is tested in a high power setup delivering 360W of stable signal power. Multiple testing degrades the fiber and transverse modal instability threshold from >360W to ~290W....

  20. IMPACT CONICAL ROD ON HARD LIMITER

    Directory of Open Access Journals (Sweden)

    Ulitin G.

    2014-12-01

    Full Text Available The problem is considered of longitudinal impact conical rod in article. A recommendation on the use of the approximate method of calculation is based on an analysis of the influence of design parameters on the value of the main oscillation frequency. There was obtained an equation of the displacement and stress of the rod. Engineering dependence has been proposed to determine the maximum force in the impact section.

  1. Self-diagnosing braided composite rod

    OpenAIRE

    Fangueiro, Raúl; Zdraveva, E.; Pereira, Cristiana Gonilho; Ferreira, A; Lanceros-Méndez, S.

    2010-01-01

    This paper presents the development of a braided reinforced composite rod (BCR) able to both reinforce and monitor the stress state of concrete structures. Carbon fibers have been used as sensing and reinforcing materials along with glass fiber. Various composites rods have been produced using an author patented technique based on a modified conventional braiding machine. The materials investigated were prepared with different carbon fiber content as follows: BCR2 (77% glass/23...

  2. Synthesis of docosahexaenoic acid from eicosapentaenoic acid in retina neurons protects photoreceptors from oxidative stress.

    Science.gov (United States)

    Simón, María Victoria; Agnolazza, Daniela L; German, Olga Lorena; Garelli, Andrés; Politi, Luis E; Agbaga, Martin-Paul; Anderson, Robert E; Rotstein, Nora P

    2016-03-01

    Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here, we investigated whether eicosapentaenoic acid (EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat and hydrogen peroxide (H2 O2 ). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase (FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in retina photoreceptors, and its precursor, eicosapentaenoic acid (EPA) have multiple beneficial effects. Here, we show that retina neurons in vitro express the desaturase FADS2 and can synthesize DHA from EPA. Moreover, addition of EPA to these cultures protects photoreceptors from oxidative stress and promotes their differentiation through its metabolization to DHA.

  3. Measurements of control rod efficiency in RBMK critical assembly upon dropping of the rods

    Energy Technology Data Exchange (ETDEWEB)

    Zhitarev, V. E., E-mail: vejitarev@nnrd.kiae.su; Kachanov, V. M.; Sergevnin, A. Yu.; Lebedev, G. V., E-mail: lgv2004@mail.ru [National Research Center Kurchatov Institute (Russian Federation)

    2014-12-15

    The efficiency of control rods in the RBMK critical assembly was measured in the case where one manual-control rod (MCR) is dropped from a steady critical state, and several other MCRs were additionally dropped after 44 s. The measured number of neutrons in the assembly during and after dropping of the rods was used to calculate the efficiency values of the rods by solution of the system of point kinetics equations. A series of methods of the initial data treatment for determination of the desired values of reactivity without the calculated corrections were used.

  4. Measurements of control rod efficiency in RBMK critical assembly upon dropping of the rods

    Science.gov (United States)

    Zhitarev, V. E.; Kachanov, V. M.; Sergevnin, A. Yu.; Lebedev, G. V.

    2014-12-01

    The efficiency of control rods in the RBMK critical assembly was measured in the case where one manual-control rod (MCR) is dropped from a steady critical state, and several other MCRs were additionally dropped after 44 s. The measured number of neutrons in the assembly during and after dropping of the rods was used to calculate the efficiency values of the rods by solution of the system of point kinetics equations. A series of methods of the initial data treatment for determination of the desired values of reactivity without the calculated corrections were used.

  5. High-throughput rod-induced electrospinning

    Science.gov (United States)

    Wu, Dezhi; Xiao, Zhiming; Teh, Kwok Siong; Han, Zhibin; Luo, Guoxi; Shi, Chuan; Sun, Daoheng; Zhao, Jinbao; Lin, Liwei

    2016-09-01

    A high throughput electrospinning process, directly from flat polymer solution surfaces induced by a moving insulating rod, has been proposed and demonstrated. Different rods made of either phenolic resin or paper with a diameter of 1-3 cm and a resistance of about 100-500 MΩ, has been successfully utilized in the process. The rod is placed approximately 10 mm above the flat polymer solution surface with a moving speed of 0.005-0.4 m s-1 this causes the solution to generate multiple liquid jets under an applied voltage of 15-60 kV for the tip-less electrospinning process. The local electric field induced by the rod can boost electrohydrodynamic instability in order to generate Taylor cones and liquid jets. Experimentally, it is found that a large rod diameter and a small solution-to-rod distance can enhance the local electrical field to reduce the magnitude of the applied voltage. In the prototype setup with poly (ethylene oxide) polymer solution, an area of 5 cm  ×  10 cm and under an applied voltage of 60 kV, the maximum throughput of nanofibers is recorded to be approximately144 g m-2 h-1.

  6. Rigid rod anchored to infinite membrane.

    Science.gov (United States)

    Guo, Kunkun; Qiu, Feng; Zhang, Hongdong; Yang, Yuliang

    2005-08-15

    We investigate the shape deformation of an infinite membrane anchored by a rigid rod. The density profile of the rod is calculated by the self-consistent-field theory and the shape of the membrane is predicted by the Helfrich membrane elasticity theory [W. Helfrich, Z. Naturforsch. 28c, 693 (1973)]. It is found that the membrane bends away from the rigid rod when the interaction between the rod and the membrane is repulsive or weakly attractive (adsorption). However, the pulled height of the membrane at first increases and then decreases with the increase of the adsorption strength. Compared to a Gaussian chain with the same length, the rigid rod covers much larger area of the membrane, whereas exerts less local entropic pressure on the membrane. An evident gap is found between the membrane and the rigid rod because the membrane's curvature has to be continuous. These behaviors are compared with that of the flexible-polymer-anchored membranes studied by previous Monte Carlo simulations and theoretical analysis. It is straightforward to extend this method to more complicated and real biological systems, such as infinite membrane/multiple chains, protein inclusion, or systems with phase separation.

  7. Long-Rod Moving-Plate Interaction

    Science.gov (United States)

    Partom, Y.

    2002-07-01

    Understanding the mechanics of interaction of a long rod projectile with a forward moving plate at an angle is essential to understanding long rod interaction with an explosive reactive armor cassette. To investigate the mechanics of such an interaction we use AUTODIN2D/EULER in plane geometry, although the problem is 3D. We assume that this is a satisfactory approximation, as we're only interested in the main features, and are not comparing fine details to experimental results. From the simulations we learn that the interaction never reaches steady state. Initially each material splits into two streams, and the interaction plane is perpendicular to the rod. But with time the interaction plane rotates slowly, until it becomes parallel to the rod, which is then able to continue moving forward without interruption. During this process interacting rod material of length DeltaL is diverted at an angle and becomes ineffective for penetrating the main target. We made many such runs to determine the dependence of DeltaL on the parameters of the problem. This dependence makes it possible to predict DeltaL for a variety of rod-plate situations.

  8. Topological optimisation of rod-stirring devices

    CERN Document Server

    Finn, Matthew D

    2011-01-01

    There are many industrial situations where rods are used to stir a fluid, or where rods repeatedly stretch a material such as bread dough or taffy. The goal in these applications is to stretch either material lines (in a fluid) or the material itself (for dough or taffy) as rapidly as possible. The growth rate of material lines is conveniently given by the topological entropy of the rod motion. We discuss the problem of optimising such rod devices from a topological viewpoint. We express rod motions in terms of generators of the braid group, and assign a cost based on the minimum number of generators needed to write the braid. We show that for one cost function -- the topological entropy per generator -- the optimal growth rate is the logarithm of the golden ratio. For a more realistic cost function,involving the topological entropy per operation where rods are allowed to move together, the optimal growth rate is the logarithm of the silver ratio, $1+\\sqrt{2}$. We show how to construct devices that realise th...

  9. How rods respond to single photons: Key adaptations of a G-protein cascade that enable vision at the physical limit of perception.

    Science.gov (United States)

    Reingruber, Jürgen; Holcman, David; Fain, Gordon L

    2015-11-01

    Rod photoreceptors are among the most sensitive light detectors in nature. They achieve their remarkable sensitivity across a wide variety of species through a number of essential adaptations: a specialized cellular geometry, a G-protein cascade with an unusually stable receptor molecule, a low-noise transduction mechanism, a nearly perfect effector enzyme, and highly evolved mechanisms of feedback control and receptor deactivation. Practically any change in protein expression, enzyme activity, or feedback control can be shown to impair photon detection, either by decreasing sensitivity or signal-to-noise ratio, or by reducing temporal resolution. Comparison of mammals to amphibians suggests that rod outer-segment morphology and the molecules and mechanism of transduction may have evolved together to optimize light sensitivity in darkness, which culminates in the extraordinary ability of these cells to respond to single photons at the ultimate limit of visual perception.

  10. Integrity of the cone photoreceptor mosaic in oligocone trichromacy

    DEFF Research Database (Denmark)

    Michaelides, Michel; Rha, Jungtae; Dees, Elise W;

    2011-01-01

    Oligocone trichromacy (OT) is an unusual cone dysfunction syndrome characterized by reduced visual acuity, mild photophobia, reduced amplitude of the cone electroretinogram with normal rod responses, normal fundus appearance, and normal or near-normal color vision. It has been proposed that these...

  11. International symposium on fuel rod simulators: development and application

    Energy Technology Data Exchange (ETDEWEB)

    McCulloch, R.W. (comp.)

    1981-05-01

    Separate abstracts are included for each of the papers presented concerning fuel rod simulator operation and performance; simulator design and evaluation; clad heated fuel rod simulators and fuel rod simulators for cladding investigations; fuel rod simulator components and inspection; and simulator analytical modeling. Ten papers have previously been input to the Energy Data Base.

  12. ABCA4 gene analysis in patients with autosomal recessive cone and cone rod dystrophies.

    Science.gov (United States)

    Kitiratschky, Veronique B D; Grau, Tanja; Bernd, Antje; Zrenner, Eberhart; Jägle, Herbert; Renner, Agnes B; Kellner, Ulrich; Rudolph, Günther; Jacobson, Samuel G; Cideciyan, Artur V; Schaich, Simone; Kohl, Susanne; Wissinger, Bernd

    2008-07-01

    The ATP-binding cassette (ABC) transporters constitute a family of large membrane proteins, which transport a variety of substrates across membranes. The ABCA4 protein is expressed in photoreceptors and possibly functions as a transporter for N-retinylidene-phosphatidylethanolamine (N-retinylidene-PE), the Schiff base adduct of all-trans-retinal with PE. Mutations in the ABCA4 gene have been initially associated with autosomal recessive Stargardt disease. Subsequent studies have shown that mutations in ABCA4 can also cause a variety of other retinal dystrophies including cone rod dystrophy and retinitis pigmentosa. To determine the prevalence and mutation spectrum of ABCA4 gene mutations in non-Stargardt phenotypes, we have screened 64 unrelated patients with autosomal recessive cone (arCD) and cone rod dystrophy (arCRD) applying the Asper Ophthalmics ABCR400 microarray followed by DNA sequencing of all coding exons of the ABCA4 gene in subjects with single heterozygous mutations. Disease-associated ABCA4 alleles were identified in 20 of 64 patients with arCD or arCRD. In four of 64 patients (6%) only one mutant ABCA4 allele was detected and in 16 patients (25%), mutations on both ABCA4 alleles were identified. Based on these data we estimate a prevalence of 31% for ABCA4 mutations in arCD and arCRD, supporting the concept that the ABCA4 gene is a major locus for various types of degenerative retinal diseases with abnormalities in cone or both cone and rod function.

  13. Local Fuel Rod Crud Prediction Tool Applications

    Energy Technology Data Exchange (ETDEWEB)

    Krammen, Michael A.; Karoutas, Zeses E.; Wang, Guoqiang; Young, Michael Y

    2009-06-15

    A code system with attendant methods has been developed for modeling local fuel rod crud. This tool is used to perform the Crud Induced Localized Corrosion (CILC) risk assessment recommended by the EPRI crud and corrosion guidelines, which were developed in response to the INPO zero fuel failures by 2010 initiatives. The methodology is in production use. This paper will describe the range of problems the methodology has already been applied to and the especial pertinence to low duty fuel applications. The methodology begins with Computational Fluid Dynamics (CFD) computations over a fuel assembly grid span. The CFD results provide detailed relative variations in local heat transfer coefficient over the grid span. These very local relative variations are used to determine very local thermal hydraulic conditions over the entire axial length of every fuel rod in a reactor core over the life of the rod in reactor. The expansion using the local relative variations is currently accomplished with the HIDUTYDRV code. The very local thermal hydraulic conditions are combined with reactor coolant crud concentrations derived from EPRI BOA analysis as input to models for predicting very local fuel rod crud deposition. The reactor coolant crud concentrations are determined over each reactor cycle by reactor system wide crud mass balance calculations. The reactor coolant crud concentrations are used to calculate local crud thickness using mass transfer models which are a function of the local thermal conditions. The advanced crud deposition models also include models for calculating local crud dryout. Local crud deposition and crud dryout are strongly dependent on very local boiling or steaming, which are predicted through the translation of the CFD results. The local crud thickness and degree of local crud dryout are key factors in determining the margin or risk for local fuel rod cladding crud induced fuel failure. The development and first application of these methods was in

  14. Regulatory perspective on incomplete control rod insertions

    Energy Technology Data Exchange (ETDEWEB)

    Chatterton, M.

    1997-01-01

    The incomplete control rod insertions experienced at South Texas Unit 1 and Wolf Creek are of safety concern to the NRC staff because they represent potential precursors to loss of shutdown margin. Even before it was determined if these events were caused by the control rods or by the fuel there was an apparent correlation of the problem with high burnup fuel. It was determined that there was also a correlation between high burnup and high drag forces as well as with rod drop time histories and lack of rod recoil. The NRC staff initial actions were aimed at getting a perspective on the magnitude of the problem as far as the number of plants and the amount of fuel that could be involved, as well as the safety significance in terms of shutdown margin. As tests have been performed and data has been analyzed the focus has shifted more toward understanding the problem and the ways to eliminate it. At this time the staff`s understanding of the phenomena is that it was a combination of factors including burnup, power history and temperature. The problem appears to be very sensitive to these factors, the interaction of which is not clearly understood. The model developed by Westinghouse provides a possible explanation but there is not sufficient data to establish confidence levels and sensitivity studies involving the key parameters have not been done. While several fixes to the problem have been discussed, no definitive fixes have been proposed. Without complete understanding of the phenomena, or fixes that clearly eliminate the problem the safety concern remains. The safety significance depends on the amount of shutdown margin lost due to incomplete insertion of the control rods. Were the control rods to stick high in the core, the reactor could not be shutdown by the control rods and other means such as emergency boration would be required.

  15. Generation, purification and transplantation of photoreceptors derived from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Deepak A Lamba

    Full Text Available BACKGROUND: Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells. METHODOLOGY/PHYSICAL FINDINGS: In this report we have used a similar method to direct induced pluripotent stem cells (iPS from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers. CONCLUSIONS: This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells.

  16. A new photosensory function for simple photoreceptors, the intrinsically photoresponsive neurons of the sea slug Onchidium

    Directory of Open Access Journals (Sweden)

    Tsukasa Gotow

    2009-12-01

    Full Text Available Simple photoreceptors, namely intrinsically light-sensitive neurons without microvilli and/or cilia, have long been known to exist in the central ganglia of crayfish, Aplysia, Onchidium, and Helix. These simple photoreceptors are not only first-order photosensory cells, but also second-order neurons (interneurons, relaying several kinds of sensory synaptic inputs. Another important issue is that the photoresponses of these simple photoreceptors show very slow kinetics and little adaptation. These characteristics suggest that the simple photoreceptors of the Onchidium have a function in non-image-forming vision, different from classical eye photoreceptors used for cording dynamic images of vision. The cited literature provides evidence that the depolarizing and hyperpolarizing photoresponses of simple photoreceptors play a role in the long-lasting potentiation of synaptic transmission of excitatory and inhibitory sensory inputs, and as well as in the potentiation and the suppression of the subsequent behavioral outputs. In short, we suggest that simple photoreceptors operate in the general potentiation of synaptic transmission and subsequent motor output; i.e., they perform a new photosensory function.

  17. Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors.

    Science.gov (United States)

    Xu, Ying; An, Futing; Borycz, Jolanta A; Borycz, Janusz; Meinertzhagen, Ian A; Wang, Tao

    2015-12-01

    Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to β-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals.

  18. The cis-regulatory logic of the mammalian photoreceptor transcriptional network.

    Directory of Open Access Journals (Sweden)

    Timothy H-C Hsiau

    Full Text Available The photoreceptor cells of the retina are subject to a greater number of genetic diseases than any other cell type in the human body. The majority of more than 120 cloned human blindness genes are highly expressed in photoreceptors. In order to establish an integrative framework in which to understand these diseases, we have undertaken an experimental and computational analysis of the network controlled by the mammalian photoreceptor transcription factors, Crx, Nrl, and Nr2e3. Using microarray and in situ hybridization datasets we have produced a model of this network which contains over 600 genes, including numerous retinal disease loci as well as previously uncharacterized photoreceptor transcription factors. To elucidate the connectivity of this network, we devised a computational algorithm to identify the photoreceptor-specific cis-regulatory elements (CREs mediating the interactions between these transcription factors and their target genes. In vivo validation of our computational predictions resulted in the discovery of 19 novel photoreceptor-specific CREs near retinal disease genes. Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression. To test the generality of this rule, we used an expanded form of it as a selection filter to evolve photoreceptor CREs from random DNA sequences in silico. When fused to fluorescent reporters, these evolved CREs drove strong, photoreceptor-specific expression in vivo. This study represents the first systematic identification and in vivo validation of CREs in a mammalian neuronal cell type and lays the groundwork for a systems biology of photoreceptor transcriptional regulation.

  19. Aversive Behavior in the Nematode C. elegans Is Modulated by cGMP and a Neuronal Gap Junction Network

    Science.gov (United States)

    Krzyzanowski, Michelle C.; Wood, Jordan F.; Brueggemann, Chantal; Bowitch, Alexander; Bethke, Mary; L’Etoile, Noelle D.; Ferkey, Denise M.

    2016-01-01

    All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal’s life history and its present internal state. Based on the integration of these variables, an animal’s needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in C. elegans. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal’s internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit. PMID:27459302

  20. Bent Telescopic Rods in Patients With Osteogenesis Imperfecta.

    Science.gov (United States)

    Lee, R Jay; Paloski, Michael D; Sponseller, Paul D; Leet, Arabella I

    2016-09-01

    Telescopic rods require alignment of 2 rods to enable lengthening. A telescopic rod converts functionally into a solid rod if either rod bends, preventing proper engagement. Our goal was to characterize implant bending as a mode of failure of telescopic rods used in the treatment of osteogenesis imperfecta in children. We conducted a retrospective review of our osteogenesis imperfecta database for patients treated with intramedullary telescopic rods at our institution from 1992 through 2010 and identified 12 patients with bent rods. The 6 boys and 6 girls had an average age at the time of initial surgery of 3.1 years (range, 1.8 to 8.3 y) and a total of 51 telescoping rods. Clinic notes, operative reports, and radiographs were reviewed. The rods were analyzed for amount of lengthening, characteristics of bending, presence of cut out, or disengagement from an anchor point. Bends in the rods were characterized by their location on the implant component. The bent and straight rods were compared. Data were analyzed with the Mann-Whitney test (statistical significance set at P≤0.05). Of the 51 telescoping rods, 17 constructs (33%) bent. The average interval between surgery and rod bending was 4.0 years (range, 0.9 to 8.2 y). Before bending, 11 of 17 telescoping rods had routine follow-up radiographs for review. In 10 of the rods, bending was present when early signs of rod failure were first detected. Rod bending did not seem to be related to rod size. There was no area on the rod itself that seemed more susceptible to bending. Rod bending can be an early sign of impending rod failure. When rod bending is first noted, it may predispose the rod to other subsequent failures such as loss of proximal and distal fixation and cut out. Rod bending should be viewed as an indicator for closer monitoring of the patient and discussions regarding future need for rod exchange. Level III-retrospective review.

  1. Induction of haem oxygenase contributes to the synthesis of pro-inflammatory cytokines in re-oxygenated rat macrophages: role of cGMP.

    Science.gov (United States)

    Tamion, F; Richard, V; Lyoumi, S; Hiron, M; Bonmarchand, G; Leroy, J; Daveau, M; Thuillez, C; Lebreton, J P

    1999-05-01

    Macrophage activation and the resulting inflammatory response may be a major component of tissue injury upon hypoxia and re-oxygenation. Activation of the haem oxygenase (HO)/carbon monoxide (CO) pathway may be an important regulator of the inflammatory response, through production of cyclic 3', 5'-monophosphate (cGMP). We have assessed whether HO contributes to the increased production of the pro-inflammatory cytokines TNF-alpha and IL-6 in re-oxygenated rat peritoneal macrophages.Hypoxia/re-oxygenation markedly increased levels of HO-1 mRNA and cGMP. The increase in cGMP was reduced by the HO-1 inhibitor tin-protoporphyrin (SnPP-9) given during re-oxygenation. Hypoxia and re-oxygenation also increased IL-6 and TNF-alpha mRNA expression, as well as IL-6 and TNF-alpha concentrations in the cell supernatant. These increases were nullified by SnPP-9 and by Methylene Blue, an inhibitor of guanylate cyclase, but were not affected by L-NNA, an inhibitor of NO synthesis. The inhibitory effect of SnPP on the synthesis of cytokines was reversed by co-administration of the stable analogue of cGMP, 8-Br-cGMP. Our results indicate that activation of haem oxygenase and of the CO/cGMP pathway is a major stimulus for the synthesis and release of pro-inflammatory cytokines in re-oxygenated macrophages. This pathway may play a central role in pathological situations in which local tissue hypoxia/re-oxygenation triggers a systemic inflammatory response, for example in patients with shock.

  2. The photochemical mechanism of a B12-dependent photoreceptor protein

    Science.gov (United States)

    Kutta, Roger J.; Hardman, Samantha J. O.; Johannissen, Linus O.; Bellina, Bruno; Messiha, Hanan L.; Ortiz-Guerrero, Juan Manuel; Elías-Arnanz, Montserrat; Padmanabhan, S.; Barran, Perdita; Scrutton, Nigel S.; Jones, Alex R.

    2015-08-01

    The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.

  3. Photoreceptors and neural circuitry underlying phototaxis in insects.

    Science.gov (United States)

    Yamaguchi, Satoko; Heisenberg, Martin

    2011-01-01

    Visual behavior of insects has long been studied, but it is only recently that a wide variety of genetic tools has become available for its analysis. Perhaps the most basic visual behaviour is phototaxis, locomotion towards a source of light. It is known in many insects and has been studied for over a century but the neural network underlying it is little understood. We recently described in the fruit fly Drosophila how different photoreceptor types contribute to phototaxis. By blocking subsets of them we showed that at least four of the five types are involved. In this short review, we compare phototactic behaviour in fruit flies and other insects (especially honeybees), and discuss what is known about the underlying neural circuitry. :

  4. Axial Vibration Confinement in Nonhomogenous Rods

    Directory of Open Access Journals (Sweden)

    S. Choura

    2005-01-01

    Full Text Available A design methodology for the vibration confinement of axial vibrations in nonhomogenous rods is proposed. This is achieved by a proper selection of a set of spatially dependent functions characterizing the rod material and geometric properties. Conditions for selecting such properties are established by constructing positive Lyapunov functions whose derivative with respect to the space variable is negative. It is shown that varying the shape of the rod alone is sufficient to confine the vibratory motion. In such a case, the vibration confinement requires that the eigenfunctions be exponentially decaying functions of space, where the notion of spatial domain stability is introduced as a concept dual to that of the time domain stability. It is also shown that vibration confinement can be produced if the rod density and/or stiffness are varied with respect to the space variable while the cross-section area is kept constant. Several case studies, supporting the developed conditions imposed on the spatially dependent functions for vibration confinement in vibrating rods, are discussed. Because variation in the geometric and material properties might decrease the critical buckling loads, we also discuss the buckling problem.

  5. Wetting of a partially immersed compliant rod

    Science.gov (United States)

    Hui, Chung-Yuen; Jagota, Anand

    2016-11-01

    The force on a solid rod partially immersed in a liquid is commonly used to determine the liquid-vapor surface tension by equating the measured force required to remove the rod from the liquid to the vertical component of the liquid-vapor surface tension. Here, we study how this process is affected when the rod is compliant. For equilibrium, we enforce force and configurational energy balance, including contributions from elastic energy. We show that, in general, the contact angle does not equal that given by Young's equation. If surface stresses are tensile, the strain in the immersed part of the rod is found to be compressive and to depend only on the solid-liquid surface stress. The strain in the dry part of the rod can be either tensile or compressive, depending on a combination of parameters that we identify. We also provide results for compliant plates partially immersed in a liquid under plane strain and plane stress. Our results can be used to extract solid surface stresses from such experiments.

  6. Single Rod Vibration in Axial Flow

    Science.gov (United States)

    Weichselbaum, Noah; Wang, Shengfu; Bardet, Philippe

    2013-11-01

    Fluid structure interaction of a single rod in axial flow is a coupled dynamical system present in many application including nuclear reactors, steam generators, and towed antenna arrays. Fluid-structure response can be quantified thanks to detailed experimental data where both structure and fluid responses are recorded. Such datum deepen understanding of the physics inherent to the system and provide high-dimensionality quantitative measurements to validate coupled structural and CFD codes with various level of complexity. In this work, single rods fixed on both ends in a concentric pipe, are subjected to an axial flow with Reynolds number based on hydraulic diameter of Re =4000. Rods of varying material stiffness and diameter are utilized in the experiment resulting in a range of dimensionless U between 0.5 and 1, where U = (ρA/EI)1/2uL. Experimental measurements of the velocity field around the rod are taken with PIV from time-resolved Nd:YLF laser and a high speed CMOS camera. Three-dimensional and temporal vibration and deflection of the rod is recorded with shadowgraphy utilizing two sets of pulsed high power LED and dedicated CMOS camera. Through integration of these two diagnostics, it is possible to reconstruct the full FSI domain providing unique validation data.

  7. Dielectric rod feed for compact range reflector

    CERN Document Server

    Balabukha, Nikolay P; Shapkina, Natalia E

    2014-01-01

    A dielectric rod feed with a special radiation pattern of a tabletop form used for the compact range reflector is developed and analyzed. Application of this feed increases the size of the compact range quiet zone generated by the reflector. The feed consists of the dielectric rod made of polystyren, the rod is inserted into the circular waveguide with a corrugated flange. The waveguide is excited by the H11-mode. The rod is covered by the textolite biconical bushing and has a fluoroplastic insert in the vicinity of the bushing. Mathematical modeling was used to obtain the parameters of the feed for the optimal tabletop form of the radiation pattern. The problem of the electromagnetic radiation was solved for metal-dielectric bodies of rotation by method of integral equations with further solving of the problem of the synthesis for feed parameters. The dielectric rod feed was fabricated for the X-frequency range. Feed amplitude and phase patterns were measured in the frequency range 8.2-12.5 GHz. Presented re...

  8. Mechanisms underlying stage-1 TRPL channel translocation in Drosophila photoreceptors.

    Directory of Open Access Journals (Sweden)

    Minh-Ha Lieu

    Full Text Available BACKGROUND: TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere, TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels. METHODOLOGY/PRINCIPAL FINDINGS: We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content. CONCLUSIONS/SIGNIFICANCE: Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protein transport, and not degradation/re-synthesis. Re-localization from each stage to the rhabdomere likely involves different strategies. Since TRPL channels can translocate to stage-1 in the absence of ATP, with no major requirement of the cytoskeleton, we suggest that stage-1 translocation involves simple diffusion through the apical membrane, which may be regulated by release of a

  9. NO regulates the strength of synaptic inputs onto hippocampal CA1 neurons via NO-GC1/cGMP signalling.

    Science.gov (United States)

    Neitz, A; Mergia, E; Neubacher, U; Koesling, D; Mittmann, T

    2015-06-01

    GABAergic interneurons are the predominant source of inhibition in the brain that coordinate the level of excitation and synchronization in neuronal circuitries. However, the underlying cellular mechanisms are still not fully understood. Here we report nitric oxide (NO)/NO-GC1 signalling as an important regulatory mechanism of GABAergic and glutamatergic synaptic transmission in the hippocampal CA1 region. Deletion of the NO receptor NO-GC1 induced functional alterations, indicated by a strong reduction of spontaneous and evoked inhibitory postsynaptic currents (IPSCs), which could be compensated by application of the missing second messenger cGMP. Moreover, we found a general impairment in the strength of inhibitory and excitatory synaptic inputs onto CA1 pyramidal neurons deriving from NO-GC1KO mice. Finally, we disclosed one subpopulation of GABAergic interneurons, fast-spiking interneurons, that receive less excitatory synaptic input and consequently respond with less spike output after blockage of the NO/cGMP signalling pathway. On the basis of these and previous findings, we propose NO-GC1 as the major NO receptor which transduces the NO signal into cGMP at presynaptic terminals of different neuronal subtypes in the hippocampal CA1 region. Furthermore, we suggest NO-GC1-mediated cGMP signalling as a mechanism which regulates the strength of synaptic transmission, hence being important in gating information processing between hippocampal CA3 and CA1 region.

  10. Rod consolidation at the West Valley Demonstration Project

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, W.J.

    1986-12-01

    A rod consolidation demonstration with irradiated pressurized water reactor fuel was recently conducted by personnel from Nuclear Assurance Corporation and West Valley Nuclear Services Company at the West Valley Demonstration Project in West Valley, New York. The rod consolidation demonstration involved pulling all of the fuel rods from six fuel Assemblies. In general, the rod pulling proceeded smoothly. The highest compaction ratio attained was 1:8:1. Among the total of 1074 fuel rods were some known degraded rods (they had collapsed cladding, a result of in-reactor fuel densification), but no rods were broken or dropped during the demonstration. One aim was to gather information on the effect of rod consolidation operations on the integrity of the fuel rods during subsequent handling and storage. Another goal was to collect information on the condition and handling of intact, damaged, and failed fuel that has been in storage for an extended period. 9 refs., 8 figs., 1 tab.

  11. Magnetic switch for reactor control rod. [LMFBR

    Science.gov (United States)

    Germer, J.H.

    1982-09-30

    A magnetic reed switch assembly is described for activating an electromagnetic grapple utilized to hold a control rod in position above a reactor core. In normal operation the magnetic field of a permanent magnet is short-circuited by a magnetic shunt, diverting the magnetic field away from the reed switch. The magnetic shunt is made of a material having a Curie-point at the desired release temperature. Above that temperature the material loses its ferromagnetic properties, and the magnetic path is diverted to the reed switch which closes and short-circuits the control circuit for the control rod electro-magnetic grapple which allows the control rod to drop into the reactor core for controlling the reactivity of the core.

  12. SNAREs Interact with Retinal Degeneration Slow and Rod Outer Segment Membrane Protein-1 during Conventional and Unconventional Outer Segment Targeting.

    Science.gov (United States)

    Zulliger, Rahel; Conley, Shannon M; Mwoyosvi, Maggie L; Stuck, Michael W; Azadi, Seifollah; Naash, Muna I

    2015-01-01

    Mutations in the photoreceptor protein peripherin-2 (also known as RDS) cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification, in vitro co-expression followed by pull-down, in vivo pull-down from mouse retinas, and proximity ligation assay to identify and confirm the SNARE proteins Syntaxin 3B and SNAP-25 as novel binding partners of RDS and ROM-1. We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional (i.e., Golgi dependent) and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1.

  13. HIGH STRENGTH CONTROL RODS FOR NEUTRONIC REACTORS

    Science.gov (United States)

    Lustman, B.; Losco, E.F.; Cohen, I.

    1961-07-11

    Nuclear reactor control rods comprised of highly compressed and sintered finely divided metal alloy panticles and fine metal oxide panticles substantially uniformly distributed theretbrough are described. The metal alloy consists essentially of silver, indium, cadmium, tin, and aluminum, the amount of each being present in centain percentages by weight. The oxide particles are metal oxides of the metal alloy composition, the amount of oxygen being present in certain percentages by weight and all the oxygen present being substantially in the form of metal oxide. This control rod is characterized by its high strength and resistance to creep at elevated temperatures.

  14. Sensitivity study of control rod depletion coefficients

    OpenAIRE

    Blomberg, Joel

    2015-01-01

    This report investigates the sensitivity of the control rod depletion coefficients, Sg, to different input parameters and how this affects the accumulated 10B depletion, β. Currently the coefficients are generated with PHOENIX4, but the geometries can be more accurately simulated in McScram. McScram is used to calculate Control Rod Worth, which in turn is used to calculate Nuclear End Of Life, and Sg cannot be generated in the current version of McScram. Therefore, it is also analyzed whether...

  15. Nitric oxide participates in cold-inhibited Camellia sinensis pollen germination and tube growth partly via cGMP in vitro.

    Directory of Open Access Journals (Sweden)

    Yu-Hua Wang

    Full Text Available Nitric oxide (NO plays essential roles in many biotic and abiotic stresses in plant development procedures, including pollen tube growth. Here, effects of NO on cold stress inhibited pollen germination and tube growth in Camellia sinensis were investigated in vitro. The NO production, NO synthase (NOS-like activity, cGMP content and proline (Pro accumulation upon treatment with NO scavenger cPTIO, NOS inhibitor L-NNA, NO donor DEA NONOate, guanylate cyclase (GC inhibitor ODQ or phosphodiesterase (PDE inhibitor Viagra at 25°C (control or 4°C were analyzed. Exposure to 4°C for 2 h reduced pollen germination and tube growth along with increase of NOS-like activity, NO production and cGMP content in pollen tubes. DEA NONOate treatment inhibited pollen germination and tube growth in a dose-dependent manner under control and reinforced the inhibition under cold stress, during which NO production and cGMP content promoted in pollen tubes. L-NNA and cPTIO markedly reduced the generation of NO induced by cold or NO donor along with partly reverse of cold- or NO donor-inhibited pollen germination and tube growth. Furthermore, ODQ reduced the cGMP content under cold stress and NO donor treatment in pollen tubes. Meanwhile, ODQ disrupted the reinforcement of NO donor on the inhibition of pollen germination and tube growth under cold condition. Additionally, Pro accumulation of pollen tubes was reduced by ODQ compared with that receiving NO donor under cold or control condition. Effects of cPTIO and L-NNA in improving cold-treated pollen germination and pollen tube growth could be lowered by Viagra. Moreover, the inhibitory effects of cPTIO and L-NNA on Pro accumulation were partly reversed by Viagra. These data suggest that NO production from NOS-like enzyme reaction decreased the cold-responsive pollen germination, inhibited tube growth and reduced Pro accumulation, partly via cGMP signaling pathway in C. sinensis.

  16. Quivers For Special Fuel Rods-Disposal Of Special Fuel Rods In CASTOR V Casks

    Energy Technology Data Exchange (ETDEWEB)

    Bannani, Amin; Cebula, Wojciech; Buchmuller, Olga; Huggenberg, Roland [GNS, Essen (Germany); Helmut Kuhl [WTI, Julich (Germany)

    2015-05-15

    While GNS casks of the CASTOR family are a suitable means to transfer fuel assemblies (FA) from the NPP to an interim dry storage site, Germanys phase-out of nuclear energy has triggered the demand for an additional solution to dispose of special fuel rods (SFR), normally remaining in the fuel pond until the final shutdown of the NPP. SFR are fuel rods that had to be removed from fuel assemblies mainly due to their special condition, e. g. damages in the cladding of the fuel rods which may have occurred during reactor operations. SFR are usually stored in the spent fuel pond after they are removed from the FA. The quiver for special fuel rods features a robust yet simple design, with a high mechanical stability, a reliable leak-tightness and large safety margins for future requirements on safety analysis. The quiver for special fuel rods can be easily adapted to a large variety of different damaged fuel rods and tailored to the specific need of the customer. The quiver for special fuel rods is adaptable e.g. in length and diameter for use in other types of transport and storage casks and is applicable in other countries as well. The overall concept presented here is a first of its kind solution for the disposal of SFRs via Castor V-casks. This provides an important precondition in achieving the status 'free from nuclear fuel' of the shut down German NPPs.

  17. An expanded set of photoreceptors in the Eastern Pale Clouded Yellow butterfly, Colias erate

    NARCIS (Netherlands)

    Pirih, Primož; Arikawa, Kentaro; Stavenga, Doekele G.

    We studied the spectral and polarisation sensitivities of photoreceptors of the butterfly Colias erate by using intracellular electrophysiological recordings and stimulation with light pulses. We developed a method of response waveform comparison (RWC) for evaluating the effective intensity of the

  18. Registration of RF Plasma Radiation in Ultra-Violet Range by Solar-blind Photoreceptor

    Science.gov (United States)

    Nguyen-Kuok, Shi; Malakhov, Yury; Korotkikh, Ivan

    2016-09-01

    A spectrum response of a photoreceptor to the RF plasma radiation is determined in the present work by means of a spectrophotometer utilizing a gas-filled photoreceptor. A continuous radiation spectrum was observed in the wavelength interval of 190 - 270 nm. The photoreceptor allows measuring of absolute radiation taking into account the spectral sensitivity of the photoreceptor and the values of quantum output for the given wavelength. A continuous spectrum was observed in all three orders of magnitude of diffraction. Develop and test a technique for measuring the intensity of the plasma radiation in the UV wavelength range measured amount of discharge pulses can be used to determine the spectral sensitivity range of UV radiation receivers. Professor.

  19. Spectral sensitivity of light induced respiratory activity of photoreceptor mitochondria in the intact fly

    NARCIS (Netherlands)

    Tinbergen, J.; Stavenga, D.G.

    1987-01-01

    Fly Calliphora erythrocephala (white eyed) photoreceptors were investigated in intact, living animals by microspectrofluorometry in vivo. The fluorescence of mitochondrial flavoproteins was used to monitor transient changes in oxidative metabolism, which were induced by a test light following a stim

  20. Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace.

    Science.gov (United States)

    Jagadish, Smitha; Barnea, Gilad; Clandinin, Thomas R; Axel, Richard

    2014-08-06

    In Drosophila, the four inner photoreceptor neurons exhibit overlapping but distinct spectral sensitivities and mediate behaviors that reflect spectral preference. We developed a genetic strategy, Tango-Trace, that has permitted the identification of the connections of the four chromatic photoreceptors. Each of the four stochastically distributed chromatic photoreceptor subtypes make distinct connections in the medulla with four different TmY cells. Moreover, each class of TmY cells forms a retinotopic map in both the medulla and the lobula complex, generating four overlapping topographic maps that could carry different color information. Thus, the four inner photoreceptors transmit spectral information through distinct channels that may converge in both the medulla and lobula complex. These projections could provide an anatomic basis for color vision and may relay information about color to motion sensitive areas. Moreover, the Tango-Trace strategy we used may be applied more generally to identify neural circuits in the fly brain.

  1. Influence of dietary melatonin on photoreceptor survival in the rat retina: an ocular toxicity study.

    Science.gov (United States)

    Wiechmann, Allan F; Chignell, Colin F; Roberts, Joan E

    2008-02-01

    Previous studies have shown that melatonin treatment increases the susceptibility of retinal photoreceptors to light-induced cell death. The purpose of this study was to evaluate under various conditions the potential toxicity of dietary melatonin on retinal photoreceptors. Male and female Fischer 344 (non-pigmented) and Long-Evans (pigmented) rats were treated with daily single doses of melatonin by gavage for a period of 14 days early in the light period or early in the dark period. In another group, rats were treated 3 times per week with melatonin early in the light period, and then exposed to high intensity illumination (1000-1500 lx; HII) for 2h, and then returned to the normal cyclic lighting regime. At the end of the treatment periods, morphometric measurements of outer nuclear layer thickness (ONL; the layer containing the photoreceptor cell nuclei) were made at specific loci throughout the retinas. In male and female non-pigmented Fischer rats, melatonin administration increased the degree of photoreceptor cell death when administered during the nighttime and during the day when followed by exposure to HII. There were some modest effects of melatonin on photoreceptor cell death when administered to Fischer rats during the day or night without exposure to HII. Melatonin treatment caused increases in the degree of photoreceptor cell death when administered in the night to male pigmented Long-Evans rats, but melatonin administration during the day, either with or without exposure to HII, had little if any effect on photoreceptor cell survival. In pigmented female Long-Evans rats, melatonin administration did not appear to have significant effects on photoreceptor cell death in any treatment group. The results of this study confirm and extend previous reports that melatonin increases the susceptibility of photoreceptors to light-induced cell death in non-pigmented rats. It further suggests that during the dark period, melatonin administration alone (i.e., no

  2. Three distinct roles for notch in Drosophila R7 photoreceptor specification.

    Directory of Open Access Journals (Sweden)

    Andrew Tomlinson

    2011-08-01

    Full Text Available Receptor tyrosine kinases (RTKs and Notch (N proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev and the EGF receptor (DER to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor. By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.

  3. Modeling and simulation performance of sucker rod beam pump

    Science.gov (United States)

    Aditsania, Annisa; Rahmawati, Silvy Dewi; Sukarno, Pudjo; Soewono, Edy

    2015-09-01

    Artificial lift is a mechanism to lift hydrocarbon, generally petroleum, from a well to surface. This is used in the case that the natural pressure from the reservoir has significantly decreased. Sucker rod beam pumping is a method of artificial lift. Sucker rod beam pump is modeled in this research as a function of geometry of the surface part, the size of sucker rod string, and fluid properties. Besides its length, sucker rod string also classified into tapered and un-tapered. At the beginning of this research, for easy modeling, the sucker rod string was assumed as un-tapered. The assumption proved non-realistic to use. Therefore, the tapered sucker rod string modeling needs building. The numerical solution of this sucker rod beam pump model is computed using finite difference method. The numerical result shows that the peak of polished rod load for sucker rod beam pump unit C-456-D-256-120, for non-tapered sucker rod string is 38504.2 lb, while for tapered rod string is 25723.3 lb. For that reason, to avoid the sucker rod string breaks due to the overload, the use of tapered sucker rod beam string is suggested in this research.

  4. Modeling and simulation performance of sucker rod beam pump

    Energy Technology Data Exchange (ETDEWEB)

    Aditsania, Annisa, E-mail: annisaaditsania@gmail.com [Department of Computational Sciences, Institut Teknologi Bandung (Indonesia); Rahmawati, Silvy Dewi, E-mail: silvyarahmawati@gmail.com; Sukarno, Pudjo, E-mail: psukarno@gmail.com [Department of Petroleum Engineering, Institut Teknologi Bandung (Indonesia); Soewono, Edy, E-mail: esoewono@math.itb.ac.id [Department of Mathematics, Institut Teknologi Bandung (Indonesia)

    2015-09-30

    Artificial lift is a mechanism to lift hydrocarbon, generally petroleum, from a well to surface. This is used in the case that the natural pressure from the reservoir has significantly decreased. Sucker rod beam pumping is a method of artificial lift. Sucker rod beam pump is modeled in this research as a function of geometry of the surface part, the size of sucker rod string, and fluid properties. Besides its length, sucker rod string also classified into tapered and un-tapered. At the beginning of this research, for easy modeling, the sucker rod string was assumed as un-tapered. The assumption proved non-realistic to use. Therefore, the tapered sucker rod string modeling needs building. The numerical solution of this sucker rod beam pump model is computed using finite difference method. The numerical result shows that the peak of polished rod load for sucker rod beam pump unit C-456-D-256-120, for non-tapered sucker rod string is 38504.2 lb, while for tapered rod string is 25723.3 lb. For that reason, to avoid the sucker rod string breaks due to the overload, the use of tapered sucker rod beam string is suggested in this research.

  5. Systemic induction of NO-, redox- and cGMP signalling in the pumpkin extrafascicular phloem upon local leaf wounding

    Directory of Open Access Journals (Sweden)

    Frank eGaupels

    2016-02-01

    Full Text Available Cucurbits developed the unique extrafascicular phloem (EFP as a defensive structure against herbivorous animals. Mechanical leaf injury was previously shown to induce a systemic wound response in the EFP of pumpkin (Cucurbita maxima. Here, we demonstrate that the phloem antioxidant system and protein modifications by NO are strongly regulated during this process. Activities of the central antioxidant enzymes dehydroascorbate reductase, glutathione reductase and ascorbate reductase were rapidly down-regulated at 30 min with a second minimum at 24 h after wounding. As a consequence levels of total ascorbate and glutathione also decreased with similar bi-phasic kinetics. These results hint towards a wound-induced shift in the redox status of the EFP. Nitric oxide (NO is another important player in stress-induced redox signalling in plants. Therefore, we analysed NO-dependent protein modifications in the EFP. Six to 48 h after leaf damage total S-nitrosothiol content and protein S-nitrosylation were clearly reduced, which was contrasted by a pronounced increase in protein tyrosine nitration. Collectively, these findings suggest that NO-dependent S-nitrosylation turned into peroxynitrite-mediated protein nitration upon a stress-induced redox shift probably involving the accumulation of reactive oxygen species within the EFP. Using the biotin switch assay and anti-nitrotyrosine antibodies we identified 9 candidate S-nitrosylated and 6 candidate tyrosine-nitrated phloem proteins. The wound-responsive Phloem Protein 16-1 (PP16-1 and Cyclophilin 18 (CYP18 as well as the 26.5 kD isoform of Phloem Protein 2 (PP2 were amenable to both NO modifications and could represent important redox-sensors within the cucurbit EFP. We also found that leaf injury triggered the systemic accumulation of cyclic guanosine monophosphate (cGMP in the EFP and discuss the possible function of this second messenger in systemic NO and redox signalling within the EFP.

  6. Photobiomodulation reduces photoreceptor death and regulates cytoprotection in early states of P23H retinal dystrophy

    Science.gov (United States)

    Kirk, Diana K.; Gopalakrishnan, Sandeep; Schmitt, Heather; Abroe, Betsy; Stoehr, Michele; Dubis, Adam; Carroll, Joseph; Stone, Jonathan; Valter, Krisztina; Eells, Janis

    2013-03-01

    Irradiation by light in the far-red to near-infrared (NIR) region of the spectrum (photobiomodulation, PBM) has been demonstrated to attenuate the severity of neurodegenerative disease in experimental and clinical studies. The purpose of this study was to test the hypothesis that 670 nm PBM would protect against the loss of retinal function and improve photoreceptor survival in a rodent model of retinitis pigmentosa, the P23H transgenic rat. P23H rat pups were treated once per day with a 670 nm LED array (180 sec treatments at 50 mW/cm2; fluence 9 joules/cm2) (Quantum Devices Inc., Barneveld WI) from postnatal day (p) 16-20 or from p10-20. Sham-treated rats were restrained, but not exposed to NIR light. The status of the retina was determined at p22 by assessment of mitochondrial function, oxidative stress and cell death. In a second series of studies, retinal status was assessed at p30 by measuring photoreceptor function by ERG and retinal morphology by Spectral Domain Optical Coherence Tomography (SD-OCT). 670 nm PBM increased retinal mitochondrial cytochrome oxidase activity and upregulated the retina's production of the key mitochondrial antioxidant enzyme, MnSOD. PBM also attenuated photoreceptor cell loss and improved photoreceptor function. PBM protects photoreceptors in the developing P23H retina, by augmenting mitochondrial function and stimulating antioxidant protective pathways. Photobiomodulation may have therapeutic potential, where mitochondrial damage is a step in the death of photoreceptors.

  7. The role of the small GTPase Rap in Drosophila R7 photoreceptor specification

    Science.gov (United States)

    Mavromatakis, Yannis Emmanuel; Tomlinson, Andrew

    2012-01-01

    The Drosophila R7 photoreceptor provides an excellent model system with which to study how cells receive and “decode” signals that specify cell fate. R7 is specified by the combined actions of the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways. These pathways interact in a complex manner that includes antagonistic effects on photoreceptor specification: RTK promotes the photoreceptor fate, whereas N inhibits. Although other photoreceptors are subject to only mild N activation, R7 experiences a high-level N signal. To counter this effect and to ensure that the cell is specified as a photoreceptor, a high RTK signal is transduced in the cell. Thus, there are two levels of RTK transduction in the photoreceptors: in R7 it is high, whereas in others it is low. Here, we address how this high-level RTK signal is transduced in R7 and find that, in addition to Ras, another small GTPase, Rap, is also engaged. Thus, when N activity is high, a robust RTK signal operates that uses both Ras and Rap, but when N activity is low, only a mild RTK signal is transduced and Ras alone suffices for the purpose. PMID:22355117

  8. Anatomy of the Hesse photoreceptor cell axonal system in the central nervous system of amphioxus.

    Science.gov (United States)

    Castro, Antonio; Becerra, Manuela; Manso, María Jesús; Sherwood, Nancy M; Anadón, Ramón

    2006-01-01

    The present study reports the organization of the Hesse cell axonal system in the central nervous system of the amphioxus, with the use of a polyclonal antiserum raised against lamprey gonadotropin-releasing hormone-I (GnRH-I). In the spinal cord, the rhabdomeric photoreceptor cells of the bicellular organs were well labeled with this antibody. These cells sent smooth, straight, lateral processes that bent and became beaded as they passed ventrally and crossed to the contralateral side of the cord. There, the processes of several cells aggregated to give rise to a longitudinal fiber bundle. Beaded collaterals of these processes were directed to ventral neuropil and did not appear to contact giant Rohde cell axons. The crossed projections of the Hesse photoreceptors are compared with those of vertebrate retinal ganglion cells. Other antisera raised against GnRH weakly labeled rhabdomeric photoreceptors located dorsally in the brain, the Joseph cells. The finding that GnRH antibodies label amphioxus photoreceptor cells and axons is not definitive proof that the photoreceptors contain GnRH. Regardless of whether the antibody recognizes amphioxus GnRH, which has not yet been identified by structure, the antibody has revealed the processes of the Hesse photoreceptor cells.

  9. Spent nuclear fuel rods encapsulated in copper

    Energy Technology Data Exchange (ETDEWEB)

    Hanes, H.D.

    1984-04-01

    Using hot isostatic pressing, spent nuclear fuel rods and other radioactive wastes can be encapsulated in solid copper. The copper capsule which is formed is free of pores and cracks, and is highly resistant to attack by reducing ground waters. Such capsules should contain radioactive materials safely for hundreds of thousands of years in underground storage.

  10. Solitary waves on nonlinear elastic rods. I

    DEFF Research Database (Denmark)

    Sørensen, Mads Peter; Christiansen, Peter Leth; Lomdahl, P. S.

    1984-01-01

    Acoustic waves on elastic rods with circular cross section are governed by improved Boussinesq equations when transverse motion and nonlinearity in the elastic medium are taken into account. Solitary wave solutions to these equations have been found. The present paper treats the interaction between...

  11. ELECTRIC FIELD MEASUREMENT IN ROD-DISCONTINUED ...

    African Journals Online (AJOL)

    2014-06-30

    Jun 30, 2014 ... The used arrangement with homogeneous system is made up of a square metallic sheet ... This distance is considered positive when the rod is located ... in the case of the discontinuous earth which were defined according to ...

  12. Fabrication of preliminary fuel rods for SFR

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun Ki; Oh, Seok Jin; Ko, Young Mo; Woo, Youn Myung; Kim, Ki Hwan [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-05-15

    Metal fuels was selected for fueling many of the first reactors in the US, including the Experimental Breeder Reactor-I (EBR-I) and the Experimental Breeder Reactor-II (EBR-II) in Idaho, the FERMI-I reactor, and the Dounreay Fast Reactor (DFR) in the UK. Metallic U.Pu.Zr alloys were the reference fuel for the US Integral Fast Reactor (IFR) program. Metallic fuel has advantages such as simple fabrication procedures, good neutron economy, high thermal conductivity, excellent compatibility with a Na coolant and inherent passive safety. U-Zr-Pu alloy fuels have been used for SFR (sodium-cooled fast reactor) related to the closed fuel cycle for managing minor actinides and reducing a high radioactivity levels since the 1980s. Fabrication technology of metallic fuel for SFR has been in development in Korea as a national nuclear R and D program since 2007. For the final goal of SFR fuel rod fabrication with good performance, recently, three preliminary fuel rods were fabricated. In this paper, the preliminary fuel rods were fabricated, and then the inspection for QC(quality control) of the fuel rods was performed

  13. Brownian rod scheme in microenvironment sensing

    Directory of Open Access Journals (Sweden)

    Ian Gralinski

    2012-03-01

    Full Text Available Fluctuations of freely translating spherical particles via Brownian motion should provide inexhaustible information about the micro-environment, but is beset by the problem of particles drifting away from the venue of measurement as well as colliding with other particles. We propose a scheme here to circumvent this in which a Brownian rod that lies in proximity to a cylindrical pillar is drawn in by a tuneable attractive force from the pillar. The force is assumed to act through the centre of each body and the motion exclusive to the x-y plane. Simulation studies show two distinct states, one in which the rod is moving freely (state I and the other in which the rod contacts the cylinder surface (state II. Information about the micro-environment could be obtained by tracking the rotational diffusion coefficient Dθ populating in either of these two states. However, the magnitude of the normalized charge product in excess of 6.3x104 was found necessary for a rod of 6.81 × 0.93 μm2 (length × diameter and 10μm diameter cylindrical pillar to minimize deviation errors. It was also found that the extent of spatial sensing coverage could be controlled by varying the charge level. The conditions needed to ascertain the rotational sampling for angle determination through the Hough transform were also discussed.

  14. Piston rod seal for a Stirling engine

    Science.gov (United States)

    Shapiro, Wilbur

    1984-01-01

    In a piston rod seal for a Stirling engine, a hydrostatic bearing and differential pressure regulating valve are utilized to provide for a low pressure differential across a rubbing seal between the hydrogen and oil so as to reduce wear on the seal.

  15. Adjustable solitary waves in electroactive rods

    Science.gov (United States)

    Wang, Y. Z.; Zhang, C. L.; Dai, H.-H.; Chen, W. Q.

    2015-10-01

    This paper presents an asymptotic analysis of solitary waves propagating in an incompressible isotropic electroactive circular rod subjected to a biasing longitudinal electric displacement. Several asymptotic expansions are introduced to simplify the rod governing equations. The boundary conditions on the lateral surface of the rod are satisfied from the asymptotic point of view. In the limit of finite-small amplitude and long wavelength, a set of ten simplified one-dimensional nonlinear governing equations is established. To validate our approach and the derivation, we compare the linear dispersion relation with the one directly derived from the three-dimensional linear theory in the limit of long wavelength. Then, by the reductive perturbation method, we deduce the far-field equation (i.e. the KdV equation). Finally, the leading order of the electroelastic solitary wave solution is presented. Numerical examples are provided to show the influences of the biasing electric displacement and material constants on the solitary waves. It is found that the biasing electric displacement can modulate the velocity of solitary waves with a prescribed amplitude in the electroactive rod, a very interesting result which may promote the particular application of solitary waves in solids with multi-field coupling.

  16. On contact numbers in random rod packings

    NARCIS (Netherlands)

    Wouterse, A.; Luding, Stefan; Philipse, A.P.

    2009-01-01

    Random packings of non-spherical granular particles are simulated by combining mechanical contraction and molecular dynamics, to determine contact numbers as a function of density. Particle shapes are varied from spheres to thin rods. The observed contact numbers (and packing densities) agree well

  17. Validation Test of CARR Safety Rod Driving Mechanism

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    <正>CARR safety Rods are driven by hydraulic force. The safety rod driving mechanism is designed by Tsinghua University and manufactured by Shenyang LIMING factory. Two sets of the mechanism are used for the validation test.

  18. Longitudinal Vibrations of Rheological Rod With Variable Cross Section

    Institute of Scientific and Technical Information of China (English)

    Katica(Stevanovic)HEDRIH; AleksandarFILIPOVSKI

    1999-01-01

    Longitudinal vibrations of rheological rod with variable cross section are examined.Particular solutions and eigenfunction are accomplished for natural vibrations of the rod with hereditary material of standard hereditary body.Some examples are given.

  19. Computer simulation of rod-sphere mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Antypov, Dmytro

    2003-07-01

    Results are presented from a series of simulations undertaken to investigate the effect of adding small spherical particles to a fluid of rods which would otherwise represent a liquid crystalline (LC) substance. Firstly, a bulk mixture of Hard Gaussian Overlap particles with an aspect ratio of 3:1 and hard spheres with diameters equal to the breadth of the rods is simulated at various sphere concentrations. Both mixing-demixing and isotropic-nematic transition are studied using Monte Carlo techniques. Secondly, the effect of adding Lennard-Jones particles to an LC system modelled using the well established Gay-Berne potential is investigated. These rod-sphere mixtures are simulated using both the original set of interaction parameters and a modified version of the rod-sphere potential proposed in this work. The subject of interest is the internal structure of the binary mixture and its dependence on density, temperature, concentration and various parameters characterising the intermolecular interactions. Both the mixing-demixing behaviour and the transitions between the isotropic and any LC phases have been studied for four systems which differ in the interaction potential between unlike particles. A range of contrasting microphase separated structures including bicontinuous, cubic, and micelle-like arrangement have been observed in bulk. Thirdly, the four types of mixtures previously studied in bulk are subjected to a static magnetic field. A variety of novel phases are observed for the cases of positive and negative anisotropy in the magnetic susceptibility. These include a lamellar structure, in which layers of rods are separated by layers of spheres, and a configuration with a self-assembling hexagonal array of spheres. Finally, two new models are presented to study liquid crystal mixtures in the presence of curved substrates. These are implemented for the cases of convex and concave spherical surfaces. The simulation results obtained in these geometries

  20. Control rod reactivity measurement by rod-drop method at a fast critical assembly

    Energy Technology Data Exchange (ETDEWEB)

    Song, L.; Yin, Y.; Lian, X.; Zheng, C. [Inst. of Nuclear Physics and Chemistry in CAEP, P. O. Box 919 210, Mianyang, Sichuan, 621900 (China)

    2012-07-01

    Rod-drop experiments were carried out to estimate the reactivity of the control rod of a fast critical assembly operated by CAEP. Two power monitor systems were used to obtain the power level and integration method was used to process the data. Three experiments were performed. The experimental results of the reactivity from the two power monitor systems were consistent and showed a reasonable range of reactivity compared to results from positive period method. (authors)

  1. Dependence of control rod worth on fuel burnup

    Energy Technology Data Exchange (ETDEWEB)

    Savva, P., E-mail: savvapan@ipta.demokritos.g [NCSR ' DEMOKRITOS' , PoB 60228, 15310 Aghia Paraskevi (Greece); Varvayanni, M., E-mail: melina@ipta.demokritos.g [NCSR ' DEMOKRITOS' , PoB 60228, 15310 Aghia Paraskevi (Greece); Catsaros, N., E-mail: nicos@ipta.demokritos.g [NCSR ' DEMOKRITOS' , PoB 60228, 15310 Aghia Paraskevi (Greece)

    2011-02-15

    Research highlights: Diffusion and MC calculations for rod worth dependence on burnup and Xe in reactors. One-step rod withdrawal/insertion are used for rod worth estimation. The study showed that when Xe is present the rods worth is significantly reduced. Rod worth variation with burnup depends on rod position in core. Rod worth obtained with MC code is higher than that obtained from deterministic. - Abstract: One important parameter in the design and the analysis of a nuclear reactor core is the reactivity worth of the control rods, i.e. their efficiency to absorb excess reactivity. The control rod worth is affected by parameters such as the fuel burnup in the rod vicinity, the Xe concentration in the core, the operational time of the rod and its position in the core. In the present work, two different computational approaches, a deterministic and a stochastic one, were used for the determination of the rods worth dependence on the fuel burnup level and the Xe concentration level in a conceptual, symmetric reactor core, based on the MTR fuel assemblies used in the Greek Research Reactor (GRR-1). For the deterministic approach the neutronics code system composed by the SCALE modules NITAWL and XSDRN and the diffusion code CITATION was used, while for the stochastic one the Monte Carlo code TRIPOLI was applied. The study showed that when Xe is present in the core, the rods worth is significantly reduced, while the rod worth variation with increasing burnup depends on the rods position in the core grid. The rod worth obtained with the use of the Monte Carlo code is higher than the one obtained from the deterministic code.

  2. Sucker rod string design of the pumping systems

    Directory of Open Access Journals (Sweden)

    Chun Hua Liu

    2015-08-01

    Full Text Available The existing design of sucker rod string mainly focuses on the simplifying assumptions that rod string was exposed to simple tension loading. And its goal was to have equal modified stress at the top of each taper. The improved rod design was to have the same degree of safety at each section, and it used a dynamic force distribution that was proportional along the whole string. However, the available procedures did not provide the desired accuracy of its pertinent analysis, and the operators could not identify the specific phenomena that occur in CBM wells. In this paper, the mathematical models of rod loads and string length were developed based on the cyclic nature of rod string loading; the fatigue endurance method is used to design the single rod string; and the tapered rod string is designed to have an equal equivalent stress at the top of each section. Its application characteristics are demonstrated by the example of CBM wells in Ordos Basin. The interpretations of results show that the previous design gave the single rods a larger diameter and the top rods in the string a greater percent than the proposed method. The calculation should concern about inertial, vibration and friction forces to illustrate the elastic force waves travelling in the rod material with the speed of sound. The single string should be designed using fatigue endurance ratings due to asymmetric pulsating tension of rod loading; and the tapered string should involve a balanced design by setting the fatigue endurance at each section equal. A shorter stroke length gives a greater rod taper percentage and an increased load capacity results to an enhanced rod diameter. The rod diameter increases with the pump size and load capacity for the single string, and the rod taper percentage of the top rod strings increases with plunger diameter for the tapered string. The proposed research improves efficiency of the pumping system, assures good operating conditions, and reduces

  3. Investigation of control rod worth and nuclear end of life of BWR control rods

    Energy Technology Data Exchange (ETDEWEB)

    Magnusson, Per

    2008-01-15

    This work has investigated the Control Rod Worth (CRW) and Nuclear End of Life (NEOL) values for BWR control rods. A study of how different parameters affect NEOL was performed with the transport code PHOENIX4. It was found that NEOL, expressed in terms of {sup 10}B depletion, can be generalized beyond the conditions for which the rod is depleted, such as different power densities and void fractions, the corresponding variation in the NEOL will be about 0.2-0.4% {sup 10}B. It was also found that NEOL results for different fuel types and different fuel enrichments have a variation of about 2-3% in {sup 10}B depletion. A comparative study on NHOL and CRW was made between PHOENIX4 and the stochastic Monte Carlo code MCNP. It was found that there is a significant difference, both due to differences in the codes and to limitations in the geometrical modeling in PHOENIX4. Since MCNP is considered more physically correct, a methodology was developed to calculate the nuclear end of life of BWR control rods with MCNP. The advantages of the methodology are that it does not require other codes to perform the depletion of the absorber material, it can describe control rods of any design and it can deplete the control rod absorber material without burning the fuel. The disadvantage of the method is that is it time-consuming.

  4. Rod Has High Tensile Strength And Low Thermal Expansion

    Science.gov (United States)

    Smith, D. E.; Everton, R. L.; Howe, E.; O'Malley, M.

    1996-01-01

    Thoriated tungsten extension rod fabricated to replace stainless-steel extension rod attached to linear variable-differential transformer in gap-measuring gauge. Threads formed on end of rod by machining with special fixtures and carefully chosen combination of speeds and feeds.

  5. 77 FR 1504 - Stainless Steel Wire Rod From India

    Science.gov (United States)

    2012-01-10

    ... COMMISSION Stainless Steel Wire Rod From India Determination On the basis of the record \\1\\ developed in the... antidumping duty order on stainless steel wire rod From India would be likely to lead to continuation or... contained in USITC Publication 4300 (January 2012), entitled Stainless Steel Wire Rod From...

  6. Carbon Inverse Opal Rods for Nonenzymatic Cholesterol Detection.

    Science.gov (United States)

    Zhong, Qifeng; Xie, Zhuoying; Ding, Haibo; Zhu, Cun; Yang, Zixue; Gu, Zhongze

    2015-11-18

    Carbon inverse opal rods made from silica photonic crystal rods are used for nonenzymatic cholesterol sensing. The characteristic reflection peak originating from the physical periodic structure works as sensing signals for quantitatively estimating cholesterol concentrations. Carbon inverse opal rods work both in cholesterol standard solutions and human serum. They are suitable for practical use in clinical diagnose.

  7. Calcium-dependent potassium current in barnacle photoreceptor.

    Science.gov (United States)

    Bolsover, S R

    1981-12-01

    When barnacle lateral eye photoreceptors are depolarized to membrane potentials of 0 to +50 mV in the dark, the plot of outward current through the cell membrane against time has two distinct maxima. The first maximum occurs 5-10 ms after the depolarization began. The current then decays to a minimum at approximately 500 ms after the onset of depolarization, and then increases to a second maximum 4-6 s after the depolarization began. If depolarization is maintained, the current again decays to reach a steady value approximately 1 min after depolarization began. The increase in current to the maximum at 4-6s from the minimum at approximately 500 ms is termed the "late current." It is maximum for depolarizations to around +25 mV and is reduced in amplitude at more positive potentials. It is not observed when the membrane is depolarized to potentials more positive than +60 mV. The late current is inhibited by external cobaltous ion and external tetraethylammonium ion, and shows a requirement for external calcium ion. When the calcium-sequestering agent EGTA is injected, the late current is abolished. Illumination of a cell under voltage clamp reduces the amplitude of the late current recorded subsequently in the dark. On the basis of the voltage dependence and pharmacology of the late current, it is proposed that the current is a calcium-dependent potassium current.

  8. Eye development and photoreceptor differentiation in the cephalopod Doryteuthis pealeii.

    Science.gov (United States)

    Koenig, Kristen M; Sun, Peter; Meyer, Eli; Gross, Jeffrey M

    2016-09-01

    Photoreception is a ubiquitous sensory ability found across the Metazoa, and photoreceptive organs are intricate and diverse in their structure. Although the morphology of the compound eye in Drosophila and the single-chambered eye in vertebrates have elaborated independently, the amount of conservation within the 'eye' gene regulatory network remains controversial, with few taxa studied. To better understand the evolution of photoreceptive organs, we established the cephalopod Doryteuthis pealeii as a lophotrochozoan model for eye development. Utilizing histological, transcriptomic and molecular assays, we characterize eye formation in Doryteuthis pealeii Through lineage tracing and gene expression analyses, we demonstrate that cells expressing Pax and Six genes incorporate into the lens, cornea and iris, and the eye placode is the sole source of retinal tissue. Functional assays demonstrate that Notch signaling is required for photoreceptor cell differentiation and retinal organization. This comparative approach places the canon of eye research in traditional models into perspective, highlighting complexity as a result of both conserved and convergent mechanisms.

  9. Nrf2 protects photoreceptor cells from photo-oxidative stress induced by blue light.

    Science.gov (United States)

    Chen, Wan-Ju; Wu, Caiying; Xu, Zhenhua; Kuse, Yoshiki; Hara, Hideaki; Duh, Elia J

    2017-01-01

    Oxidative stress plays a key role in age-related macular degeneration and hereditary retinal degenerations. Light damage in rodents has been used extensively to model oxidative stress-induced photoreceptor degeneration, and photo-oxidative injury from blue light is particularly damaging to photoreceptors. The endogenous factors protecting photoreceptors from oxidative stress, including photo-oxidative stress, are continuing to be elucidated. In this study, we evaluated the effect of blue light exposure on photoreceptors and its relationship to Nrf2 using cultured murine photoreceptor (661W) cells. 661W cells were exposed to blue light at 2500 lux. Exposure to blue light for 6-24 h resulted in a significant increase in intracellular reactive oxygen species (ROS) and death of 661W cells in a time-dependent fashion. Blue light exposure resulted in activation of Nrf2, as indicated by an increase in nuclear translocation of Nrf2. This was associated with a significant induction of expression of Nrf2 as well as an array of Nrf2 target genes, including antioxidant genes, as indicated by quantitative reverse transcription PCR (qRT-PCR). In order to determine the functional role of Nrf2, siRNA-mediated knockdown studies were performed. Nrf2-knockdown in 661W cells resulted in significant exacerbation of blue light-induced reactive oxygen species levels as well as cell death. Taken together, these findings indicate that Nrf2 is an important endogenous protective factor against oxidative stress in photoreceptor cells. This suggests that drugs targeting Nrf2 could be considered as a neuroprotective strategy for photoreceptors in AMD and other retinal conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Four of the six Drosophila rhodopsin-expressing photoreceptors can mediate circadian entrainment in low light.

    Science.gov (United States)

    Saint-Charles, Alexandra; Michard-Vanhée, Christine; Alejevski, Faredin; Chélot, Elisabeth; Boivin, Antoine; Rouyer, François

    2016-10-01

    Light is the major stimulus for the synchronization of circadian clocks with day-night cycles. The light-driven entrainment of the clock that controls rest-activity rhythms in Drosophila relies on different photoreceptive molecules. Cryptochrome (CRY) is expressed in most brain clock neurons, whereas six different rhodopsins (RH) are present in the light-sensing organs. The compound eye includes outer photoreceptors that express RH1 and inner photoreceptors that each express one of the four rhodopsins RH3-RH6. RH6 is also expressed in the extraretinal Hofbauer-Buchner eyelet, whereas RH2 is only found in the ocelli. In low light, the synchronization of behavioral rhythms relies on either CRY or the canonical rhodopsin phototransduction pathway, which requires the phospholipase C-β encoded by norpA (no receptor potential A). We used norpA(P24) cry(02) double mutants that are circadianly blind in low light and restored NORPA function in each of the six types of photoreceptors, defined as expressing a particular rhodopsin. We first show that the NORPA pathway is less efficient than CRY for synchronizing rest-activity rhythms with delayed light-dark cycles but is important for proper phasing, whereas the two light-sensing pathways can mediate efficient adjustments to phase advances. Four of the six rhodopsin-expressing photoreceptors can mediate circadian entrainment, and all are more efficient for advancing than for delaying the behavioral clock. In contrast, neither RH5-expressing retinal photoreceptors nor RH2-expressing ocellar photoreceptors are sufficient to mediate synchronization through the NORPA pathway. Our results thus reveal different contributions of rhodopsin-expressing photoreceptors and suggest the existence of several circuits for rhodopsin-dependent circadian entrainment. J. Comp. Neurol. 524:2828-2844, 2016. © 2016 Wiley Periodicals, Inc.

  11. Contribution of calpains to photoreceptor cell death in N-methyl-N-nitrosourea-treated rats.

    Science.gov (United States)

    Oka, Takayuki; Nakajima, Takeshi; Tamada, Yoshiyuki; Shearer, Thomas R; Azuma, Mitsuyoshi

    2007-03-01

    The purpose of the present study was to determine if proteolysis by the calcium-dependent enzyme calpains (EC 3.4.22.17) contributed to retinal cell death in a rat model of photoreceptor degeneration induced by intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Retinal degeneration was evaluated by H&E staining, and cell death was determined by TUNEL assay. Total calcium in retina was measured by atomic absorption spectrophotometry. Activation of calpains was determined by casein zymography and immunoblotting. Proteolysis of alpha-spectrin and p35 (regulator of Cdk5) were evaluated by immunoblotting. Calpain inhibitor SNJ-1945 was orally administrated to MNU-treated rats to test drug efficacy. MNU decreased the thickness of photoreceptor cell layer, composed of the outer nuclear layer (ONL) and outer segment (OS). Numerous cells in the ONL showed positive TUNEL staining. Total calcium was increased in retina after MNU. Activation of calpains and calpain-specific proteolysis of alpha-spectrin were observed after MNU injection. Oral administration of SNJ-1945 to MNU-treated rats showed a significant protective effect against photoreceptor cell loss, confirming involvement of calpains in photoreceptor degeneration. Conversion of p35 to p25 was well correlated with calpain activation, suggesting prolonged activation of Cdk5/p25 as a possible downstream mechanism for MNU-induced photoreceptor cell death. SNJ-1945 reduced photoreceptor cells death, even though MNU is one of the most severe models of photoreceptor cell degeneration. Oral calpain inhibitor SNJ-1945 may be a candidate for testing as a medication against retinal degeneration in retinitis pigmentosa.

  12. Time-lapse imaging as a tool to investigate contractility of the epididymal duct--effects of cGMP signaling.

    Directory of Open Access Journals (Sweden)

    Andrea Mietens

    Full Text Available The well orchestrated function of epididymal smooth muscle cells ensures transit of spermatozoa through the epididymal duct during which spermatozoa acquire motility and fertilizing capacity. Relaxation of smooth muscle cells is mediated by cGMP signaling and components of this pathway are found within the male reproductive tract. Whereas contractile function of caudal parts of the rat epididymal duct can be examined in organ bath studies, caput and corpus regions are fragile and make it difficult to mount them in an organ bath. We developed an ex vivo time-lapse imaging-based approach to investigate the contractile pattern in these parts of the epididymal duct. Collagen-embedding allowed immobilization without impeding contractility or diffusion of drugs towards the duct and therefore facilitated subsequent movie analyses. The contractile pattern was made visible by placing virtual sections through the acquired image stack to track wall movements over time. By this, simultaneous evaluation of contractile activity at different positions of the observed duct segment was possible. With each contraction translating into a spike, drug-induced alterations in contraction frequency could be assessed easily. Peristaltic contractions were also detectable and throughout all regions in the proximal epididymis we found regular spontaneous contractile activity that elicited movement of intraluminal contents. Stimulating cGMP production by natriuretic peptide ANP or inhibiting degradation of cGMP by the phosphodiesterase 5 inhibitor sildenafil significantly reduced contractile frequency in isolated duct segments from caput and corpus. RT-PCR analysis after laser-capture microdissection localized the corresponding molecules to the smooth muscle layer of the duct. Our time-lapse imaging approach proved to be feasible to assess contractile function in all regions of the epididymal duct under near physiological conditions and provides a tool to evaluate acute

  13. Effects of hydrazine derivatives on vascular smooth muscle contractility, blood pressure and cGMP production in rats: comparison with hydralazine.

    Science.gov (United States)

    Vidrio, Horacio; Fernández, Gabriela; Medina, Martha; Alvarez, Ezequiel; Orallo, Francisco

    2003-01-01

    Hydralazine is a hydrazine derivative used clinically as a vasodilator and antihypertensive agent. Despite numerous studies with the drug, its mechanism of action has remained unknown; guanylate cyclase activation and release of endothelial relaxing factors are thought to be involved in its vasodilator effect. Other hydrazine derivatives are known to stimulate guanylate cyclase and could therefore share the vasodilator activity of hydralazine, although such possibility has not been assessed systematically. In the present study, hydralazine, hydrazine, phenylhydrazine, and isoniazid were evaluated for vascular smooth muscle relaxation in rat aortic rings with and without endothelium, as well as after incubation with the guanylate cyclase inhibitor methylene blue. They were also tested for enhancement of cyclic guanosine monophosphate (cGMP) production by cultured rat aortic smooth muscle cells and for hypotension in the anesthetized rat. All hydrazines relaxed aortic rings, an action unaffected by endothelium removal and, in all cases except hydralazine, antagonized by methylene blue. Only phenylhydrazine increased cGMP production and only hydralazine markedly lowered blood pressure. It was concluded that hydralazine vascular relaxation is independent of endothelium and is not related to guanylate cyclase activation. The other hydrazines studied also elicit endothelium-independent relaxation, but the effect is related to guanylate cyclase. The marked hypotensive effect of hydralazine contrasts with its modest relaxant activity and is not shared by the other hydrazines. The fact that hydrazine and isoniazid produce methylene blue-sensitive relaxation, yet do not enhance cGMP production suggests the need for activating factors present in aortic rings but not in isolated cells.

  14. Nitric oxide synthetic pathway and cGMP levels are altered in red blood cells from end-stage renal disease patients.

    Science.gov (United States)

    Di Pietro, Natalia; Giardinelli, Annalisa; Sirolli, Vittorio; Riganti, Chiara; Di Tomo, Pamela; Gazzano, Elena; Di Silvestre, Sara; Panknin, Christina; Cortese-Krott, Miriam M; Csonka, Csaba; Kelm, Malte; Ferdinandy, Péter; Bonomini, Mario; Pandolfi, Assunta

    2016-06-01

    Red blood cells (RBCs) enzymatically produce nitric oxide (NO) by a functional RBC-nitric oxide synthase (RBC-NOS). NO is a vascular key regulatory molecule. In RBCs its generation is complex and influenced by several factors, including insulin, acetylcholine, and calcium. NO availability is reduced in end-stage renal disease (ESRD) and associated with endothelial dysfunction. We previously demonstrated that, through increased phosphatidylserine membrane exposure, ESRD-RBCs augmented their adhesion to human cultured endothelium, in which NO bioavailability decreased. Since RBC-NOS-dependent NO production in ESRD is unknown, this study aimed to investigate RBC-NOS levels/activation, NO production/bioavailability in RBCs from healthy control subjects (C, N = 18) and ESRD patients (N = 27). Although RBC-NOS expression was lower in ESRD-RBCs, NO, cyclic guanosine monophosphate (cGMP), RBC-NOS Serine1177 phosphorylation level and eNOS/Calmodulin (CaM)/Heat Shock Protein-90 (HSP90) interaction levels were higher in ESRD-RBCs, indicating increased enzyme activation. Conversely, following RBCs stimulation with insulin or ionomycin, NO and cGMP levels were significantly lower in ESRD- than in C-RBCs, suggesting that uremia might reduce the RBC-NOS response to further stimuli. Additionally, the activity of multidrug-resistance-associated protein-4 (MRP4; cGMP-membrane transporter) was significantly lower in ESRD-RBCs, suggesting a possible compromised efflux of cGMP across the ESRD-RBCs membrane. This study for the first time showed highest basal RBC-NOS activation in ESRD-RBCs, possibly to reduce the negative impact of decreased NOS expression. It is further conceivable that high NO production only partially affects cell function of ESRD-RBCs maybe because in vivo they are unable to respond to physiologic stimuli, such as calcium and/or insulin.

  15. GARP2 accelerates retinal degeneration in rod cGMP-gated cation channel β-subunit knockout mice

    Science.gov (United States)

    DeRamus, Marci L.; Stacks, Delores A.; Zhang, Youwen; Huisingh, Carrie E.; McGwin, Gerald; Pittler, Steven J.

    2017-01-01

    The Cngb1 locus-encoded β-subunit of rod cGMP-gated cation channel and associated glutamic acid rich proteins (GARPs) are required for phototransduction, disk morphogenesis, and rod structural integrity. To probe individual protein structure/function of the GARPs, we have characterized several transgenic mouse lines selectively restoring GARPs on a Cngb1 knockout (X1−/−) mouse background. Optical coherence tomography (OCT), light and transmission electron microscopy (TEM), and electroretinography (ERG) were used to analyze 6 genotypes including WT at three and ten weeks postnatal. Comparison of aligned histology/OCT images demonstrated that GARP2 accelerates the rate of degeneration. ERG results are consistent with the structural analyses showing the greatest attenuation of function when GARP2 is present. Even 100-fold or more overexpression of GARP1 could not accelerate degeneration as rapidly as GARP2, and when co-expressed GARP1 attenuated the structural and functional deficits elicited by GARP2. These results indicate that the GARPs are not fully interchangeable and thus, likely have separate and distinct functions in the photoreceptor. We also present a uniform murine OCT layer naming nomenclature system that is consistent with human retina layer designations to standardize murine OCT, which will facilitate data evaluation across different laboratories. PMID:28198469

  16. RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations

    Directory of Open Access Journals (Sweden)

    Li eJiang

    2014-04-01

    Full Text Available RNA interference (RNAi knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies. To test this, we used self-complementary (sc AAV2/8 vector to develop an RNAi-based therapy in two dominant retinal degeneration mouse models. The allele-specific model expresses transgenic bovine GCAP1(Y99C establishing a rapid RP-like phenotype, whereas the nonallele-specific model expresses mouse GCAP1(L151F producing a slowly progressing cone/rod dystrophy (CORD. The late onset GCAP1(L151F-CORD mimics the dystrophy observed in human GCAP1-CORD patients. Subretinal injection of scAAV2/8 carrying shRNA expression cassettes specific for bovine or mouse GCAP1 showed strong expression at one week post-injection. In both allele-specific (GCAP1(Y99C-RP and nonallele-specific (GCAP1(L151F-CORD models of dominant retinal dystrophy, RNAi-mediated gene silencing enhanced photoreceptor survival, delayed onset of degeneration and improved visual function. Such results provide a proof of concept toward effective RNAi-based gene therapy mediated by scAAV2/8 for dominant retinal disease based on GCAP1 mutation. Further, nonallele-specific RNAi knockdown of GCAP1 may prove generally applicable toward the rescue of any human GCAP1-based dominant cone-rod dystrophy.

  17. STUDY ON A HYDROPHOBIC-HYDROPHILIC GRADIENT ROD

    Institute of Scientific and Technical Information of China (English)

    Jun Ma; Bai-yu Li; Hai-yun Liu; Zhi-min Zheng; Jian Xu

    2004-01-01

    A hydrophobic-hydrophilic gradient rod with a length of 40 mm and a diameter of 3 mm was prepared by heating a polymethylsilsesquioxane rod in a cylindrical stove with temperature gradient. The rod was thus pyrolyzed under a temperature gradient condition. The organic end of the gradient rod appears hydrophobic with a contact angle of 109.9° while the other end is hydrophilic with a contact angle of 62.4°. The gradient chemical structure and the gradient microstructure along the rod were characterized by FTIR and SEM, respectively.

  18. Test Research on Special Sucker Rod for Screw Pump

    Institute of Scientific and Technical Information of China (English)

    Zhang Mingyi; Chen Mingzhan; Li Zhi

    2006-01-01

    @@ According to the statistics of straight thread sucker rods' application in screw pump in Daqing Oilfield before2000, the proportion of sucker rods' yearly breakaway reached to 41.6%, taking up 70% of the total wells that were checked. Thus it can be seen that the rods breakaway problem was becoming the main barrier restricting screw pump large-scale population and application. Since then,the development work on the special sucker rods for screw pump had been carried on. Through the analysis on the failure position and failure form of the sucker rods',the following conclusions arepresented:

  19. Photonic mesophases from cut rod rotators

    Energy Technology Data Exchange (ETDEWEB)

    Stelson, Angela C.; Liddell Watson, Chekesha M., E-mail: cml66@cornell.edu [Materials Science and Engineering, Cornell University, Ithaca, New York 14853 (United States); Avendano, Carlos [Chemical Engineering and Analytical Science, The University of Manchester, Manchester M13 9PL (United Kingdom)

    2016-01-14

    The photonic band properties of random rotator mesophases are calculated using supercell methods applied to cut rods on a hexagonal lattice. Inspired by the thermodynamic mesophase for anisotropic building blocks, we vary the shape factor of cut fraction for the randomly oriented basis. We find large, stable bandgaps with high gap isotropy in the inverted and direct structures as a function of cut fraction, dielectric contrast, and filling fraction. Bandgap sizes up to 34.5% are maximized at high dielectric contrast for rods separated in a matrix. The bandgaps open at dielectric contrasts as low as 2.0 for the transverse magnetic polarization and 2.25 for the transverse electric polarization. Additionally, the type of scattering that promotes the bandgap is correlated with the effect of disorder on bandgap size. Slow light properties are investigated in waveguide geometry and slowdown factors up to 5 × 10{sup 4} are found.

  20. Oligo(naphthylene–ethynylene) Molecular Rods

    DEFF Research Database (Denmark)

    Cramer, Jacob Roland; Ning, Yanxiao; Shen, Cai;

    2013-01-01

    Molecular rods designed for surface chirality studies have been synthesized in high yields. The molecules are composed of oligo(naphthylene–ethynylene) skeletons and functionalized at their two termini with carboxylic acids and hydrophobic groups. The molecular skeletons were constructed by means...... of palladium-catalyzed Sonogashira reactions between naphthyl halides and acetylenes. The triazene functionality was used as a protected iodine precursor to allow linear extension of the molecular rods during the synthe-ses. The carboxylic acid groups in the target molecules were protected as esters during...... the synthesis to keep the large aromatic molecules soluble during their syntheses. These rigid oligomers were designed to form lamella-like structures when adsorbed on a surface, through which multiple distinguishable surface conformations should be obtainable. Preliminary scanning tunneling microscopy imaging...

  1. [Rod of Asclepius. Symbol of medicine].

    Science.gov (United States)

    Young, Pablo; Finn, Bárbara C; Bruetman, Julio E; Cesaro Gelos, Jorge; Trimarchi, Hernán

    2013-09-01

    Symbolism is one of the most archaic forms of human thoughts. Symbol derives from the Latin word symbolum, and the latter from the Greek symbolon or symballo, which means "I coincide, I make matches". The Medicine symbol represents a whole series of historical and ethical values. Asclepius Rod with one serpent entwined, has traditionally been the symbol of scientific medicine. In a misconception that has lasted 500 years, the Caduceus of Hermes, entwined by two serpents and with two wings, has been considered the symbol of Medicine. However, the Caduceus is the current symbol of Commerce. Asclepius Rod and the Caduceus of Hermes represent two professions, Medicine and Commerce that, in ethical practice, should not be mixed. Physicians should be aware of their real emblem, its historical origin and meaning.

  2. Composites reinforcement by rods: a SAS study

    Energy Technology Data Exchange (ETDEWEB)

    Urban, V. [ESRF, BP220, 38043 Grenoble Cedex (France); Botti, A.; Pyckhout-Hintzen, W.; Richter, D. [IFF-Forschungszentrum Juelich, 52425 Juelich (Germany); Straube, E. [University of Halle, FB Physik, 06099 Halle (Germany)

    2002-07-01

    The mechanical properties of composites are governed by size, shape and dispersion degree of so-called reinforcing particles. Polymeric fillers based on thermodynamically driven microphase separation of block copolymers offer the opportunity to study a model system of controlled rod-like filler particles. We chose a triblock copolymer (PBPSPB) and carried out SAS measurements with both X-rays and neutrons, in order to characterize separately the hard phase and the cross-linked PB matrix. The properties of the material depend strongly on the way that stress is carried and transferred between the soft matrix and the hard fibers. The failure of the strain-amplification concept and the change of topological contributions to the free energy and scattering factor have to be addressed. In this respect the composite shows a similarity to a two-network system, i.e. interpenetrating rubber and rod-like filler networks. (orig.)

  3. Composites reinforcement by rods a SAS study

    CERN Document Server

    Urban, V; Pyckhout-Hintzen, W; Richter, D; Straube, E

    2002-01-01

    The mechanical properties of composites are governed by size, shape and dispersion degree of so-called reinforcing particles. Polymeric fillers based on thermodynamically driven microphase separation of block copolymers offer the opportunity to study a model system of controlled rod-like filler particles. We chose a triblock copolymer (PBPSPB) and carried out SAS measurements with both X-rays and neutrons, in order to characterize separately the hard phase and the cross-linked PB matrix. The properties of the material depend strongly on the way that stress is carried and transferred between the soft matrix and the hard fibers. The failure of the strain-amplification concept and the change of topological contributions to the free energy and scattering factor have to be addressed. In this respect the composite shows a similarity to a two-network system, i.e. interpenetrating rubber and rod-like filler networks. (orig.)

  4. Instabilities of a rotating helical rod

    Science.gov (United States)

    Park, Yunyoung; Ko, William; Kim, Yongsam; Lim, Sookkyung

    2016-11-01

    Bacteria such as Escherichia coli and Vibrio alginolyticus have helical flagellar filament. By rotating a motor, which is located at the bottom end of the flagellar filament embedded in the cell body, CCW or CW, they swim forward or backward. We model a left-handed helix by the Kirchhoff rod theory and use regularized Stokes formulation to study an interaction between the surrounding fluid and the flagellar filament. We perform numerical studies focusing on relations between physical parameters and critical angular frequency of the motor, which separates overwhiring from twirling. We are also interested in the buckling instability of the hook, which is very flexible elastic rod. By measuring buckling angle, which is an angle between rotational axis and helical axis, we observe the effects of physical parameters on buckling of the hook.

  5. Hollow Sucker Rod Applied in Production Engineering

    Institute of Scientific and Technical Information of China (English)

    Wang Tongbin; Liu Liandong; Hu Daoming; Jia Yanshan

    1997-01-01

    @@ Working Principle A positive cycle system or a working channel can be formed by means of hollow sucker rod and its mating parts in the oil tube ofa well, through which heat carriers (such as hot water,hot oil and steam), chemicals and heating cable can be pumped or put into the well so as to lower the viscosity of crude, dissolve the paraffin building-up and open the conduit, thus leading to the smooth oil flow out of well.

  6. Rod Driven Frequency Entrainment and Resonance Phenomena

    Directory of Open Access Journals (Sweden)

    Christina Salchow

    2016-08-01

    Full Text Available A controversy exists on photic driving in the human visual cortex evoked by intermittent photic stimulation. Frequency entrainment and resonance phenomena are reported for frequencies higher than 12 Hz in some studies while missing in others. We hypothesized that this might be due to different experimental conditions, since both high and low intensity light stimulation were used. However, most studies do not report radiometric measurements, which makes it impossible to categorize the stimulation according to photopic, mesopic, and scotopic vision. Low intensity light stimulation might lead to scotopic vision, where rod perception dominates. In this study, we investigated photic driving for rod-dominated visual input under scotopic conditions. Twelve healthy volunteers were stimulated with low intensity light flashes at 20 stimulation frequencies, leading to rod activation only. The frequencies were multiples of the individual alpha frequency (α of each volunteer in the range from 0.40–2.30*α. 306-channel whole head magnetoencephalography recordings were analyzed in time, frequency, and spatiotemporal domains with the Topographic Matching Pursuit algorithm. We found resonance phenomena and frequency entrainment for stimulations at or close to the individual alpha frequency (0.90–1.10*α and half of the alpha frequency (0.40–0.55*α. No signs of resonance and frequency entrainment phenomena were revealed around 2.00*α. Instead, on-responses at the beginning and off-responses at the end of each stimulation train were observed for the first time in a photic driving experiment at frequencies of 1.30–2.30*α, indicating that the flicker fusion threshold was reached. All results, the resonance and entrainment as well as the fusion effects, provide evidence for rod-dominated photic driving in the visual cortex.

  7. Rod Driven Frequency Entrainment and Resonance Phenomena

    Science.gov (United States)

    Salchow, Christina; Strohmeier, Daniel; Klee, Sascha; Jannek, Dunja; Schiecke, Karin; Witte, Herbert; Nehorai, Arye; Haueisen, Jens

    2016-01-01

    A controversy exists on photic driving in the human visual cortex evoked by intermittent photic stimulation. Frequency entrainment and resonance phenomena are reported for frequencies higher than 12 Hz in some studies while missing in others. We hypothesized that this might be due to different experimental conditions, since both high and low intensity light stimulation were used. However, most studies do not report radiometric measurements, which makes it impossible to categorize the stimulation according to photopic, mesopic, and scotopic vision. Low intensity light stimulation might lead to scotopic vision, where rod perception dominates. In this study, we investigated photic driving for rod-dominated visual input under scotopic conditions. Twelve healthy volunteers were stimulated with low intensity light flashes at 20 stimulation frequencies, leading to rod activation only. The frequencies were multiples of the individual alpha frequency (α) of each volunteer in the range from 0.40 to 2.30∗α. Three hundred and six-channel whole head magnetoencephalography recordings were analyzed in time, frequency, and spatiotemporal domains with the Topographic Matching Pursuit algorithm. We found resonance phenomena and frequency entrainment for stimulations at or close to the individual alpha frequency (0.90–1.10∗α) and half of the alpha frequency (0.40–0.55∗α). No signs of resonance and frequency entrainment phenomena were revealed around 2.00∗α. Instead, on-responses at the beginning and off-responses at the end of each stimulation train were observed for the first time in a photic driving experiment at frequencies of 1.30–2.30∗α, indicating that the flicker fusion threshold was reached. All results, the resonance and entrainment as well as the fusion effects, provide evidence for rod-dominated photic driving in the visual cortex. PMID:27588002

  8. Pupillary Light Reflexes in Severe Photoreceptor Blindness Isolate the Melanopic Component of Intrinsically Photosensitive Retinal Ganglion Cells

    Science.gov (United States)

    Charng, Jason; Jacobson, Samuel G.; Heon, Elise; Roman, Alejandro J.; McGuigan, David B.; Sheplock, Rebecca; Kosyk, Mychajlo S.; Swider, Malgorzata; Cideciyan, Artur V.

    2017-01-01

    Purpose Pupillary light reflex (PLR) is driven by outer retinal photoreceptors and by melanopsin-expressing intrinsically photosensitive retinal ganglion cells of the inner retina. To isolate the melanopic component, we studied patients with severe vision loss due to Leber congenital amaurosis (LCA) caused by gene mutations acting on the outer retina. Methods Direct PLR was recorded in LCA patients (n = 21) with known molecular causation and severe vision loss. Standard stimuli (2.5 log scot-cd.m−2; ∼13 log quanta.cm−2.s−1; achromatic full-field) with 0.1- or 5-second duration were used in all patients. Additional recordings were performed with higher luminance (3.9 log scot-cd.m−2) in a subset of patients. Results The LCA patients showed no detectable PLR to the standard stimulus with short duration. With longer-duration stimuli, a PLR was detectable in the majority (18/21) of patients. The latency of the PLR was 2.8 ± 1.3 seconds, whereas normal latency was 0.19 ± 0.02 seconds. Peak contraction amplitude in patients was 1.1 ± 0.9 mm at 6.2 ± 2.3 seconds, considerably different from normal amplitude of 4.2 ± 0.4 mm at 3.0 ± 0.4 seconds. Recordings with higher luminance demonstrated that PLRs in severe LCA could also be evoked with short-duration stimuli. Conclusions The PLR in severe LCA patients likely represents the activation of the melanopic circuit in isolation from rod and cone input. Knowledge of the properties of the human melanopic PLR allows not only comparison to those in animal models but also serves to define the fidelity of postretinal transmission in clinical trials targeting patients with no outer retinal function. PMID:28660274

  9. Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography.

    Science.gov (United States)

    Nickell, Stephan; Park, Paul S-H; Baumeister, Wolfgang; Palczewski, Krzysztof

    2007-06-01

    The rod outer segment (ROS) of photoreceptor cells houses all components necessary for phototransduction, a set of biochemical reactions that amplify and propagate a light signal. Theoretical approaches to quantify this process require precise information about the physical boundaries of the ROS. Dimensions of internal structures within the ROS of mammalian species have yet to be determined with the precision required for quantitative considerations. Cryoelectron tomography was utilized to obtain reliable three-dimensional morphological information about this important structure from murine retina. Vitrification of samples permitted imaging of the ROS in a minimally perturbed manner and the preservation of substructures. Tomograms revealed the characteristic highly organized arrangement of disc membranes stacked on top of one another with a surrounding plasma membrane. Distances among the various membrane components of the ROS were measured to define the space available for phototransduction to occur. Reconstruction of segments of the ROS from single-axis tilt series images provided a glimpse into the three-dimensional architecture of this highly differentiated neuron. The reconstructions revealed spacers that likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs.

  10. ELECTROMAGNETIC APPARATUS FOR MOVING A ROD

    Science.gov (United States)

    Young, J.N.

    1957-08-20

    An electromagnetic device for moving an object in a linear path by increments is described. The device is specifically adapted for moving a neutron absorbing control rod into and out of the core of a reactor and consists essentially of an extension member made of magnetic material connected to one end of the control rod and mechanically flexible to grip the walls of a sleeve member when flexed, a magnetic sleeve member coaxial with and slidable between limit stops along the flexible extension, electromagnetic coils substantially centrally located with respect to the flexible extension to flex the extension member into gripping engagement with the sleeve member when ener gized, moving electromagnets at each end of the sleeve to attract the sleeve when energized, and a second gripping electromagnet positioned along the flexible extension at a distance from the previously mentioned electromagnets for gripping the extension member when energized. In use, the second gripping electromagnet is deenergized, the first gripping electromagnet is energized to fix the extension member in the sleeve, and one of the moving electromagnets is energized to attract the sleeve member toward it, thereby moving the control rod.

  11. Description and characterization of HBWR Series H-1 test rods

    Energy Technology Data Exchange (ETDEWEB)

    Wagoner, S.R.; Barner, J.O.; Welty, R.K.

    1979-06-01

    The as-built characterization results are presented for the HBWR Series H-1 test rods to be irradiated as part of the Fuel Performance Improvement Program (FPIP). The irradiation of these rods is to be conducted in the Halden Boiling Water Reactor (HBWR). Series H-1 consists of twelve rods for irradiation and six spares. Rod design types include (1) a reference dished pellet design, (2) an annular pellet design, (3) an annular pellet design combined with graphite-coated cladding, and (4) a packed-particle (vipac) design. The report, which describes the fabrication and detailed characterization results for the rods, is divided into four major sections: (1) experiment description, (2) process development required to fabricate the test rods, (3) methods and procedures used to fabricate and characterize the rods, and (4) a summary of the characterization results.

  12. Nuclear thermionic converter. [tungsten-thorium oxide rods

    Science.gov (United States)

    Phillips, W. M.; Mondt, J. F. (Inventor)

    1977-01-01

    Efficient nuclear reactor thermionic converter units are described which can be constructed at low cost and assembled in a reactor which requires a minimum of fuel. Each converter unit utilizes an emitter rod with a fluted exterior, several fuel passages located in the bulges that are formed in the rod between the flutes, and a collector receiving passage formed through the center of the rod. An array of rods is closely packed in an interfitting arrangement, with the bulges of the rods received in the recesses formed between the bulges of other rods, thereby closely packing the nuclear fuel. The rods are constructed of a mixture of tungsten and thorium oxide to provide high power output, high efficiency, high strength, and good machinability.

  13. Optical properties of photoreceptor and retinal pigment epithelium cells investigated with adaptive optics optical coherence tomography

    Science.gov (United States)

    Liu, Zhuolin

    Human vision starts when photoreceptors collect and respond to light. Photoreceptors do not function in isolation though, but share close interdependence with neighboring photoreceptors and underlying retinal pigment epithelium (RPE) cells. These cellular interactions are essential for normal function of the photoreceptor-RPE complex, but methods to assess these in the living human eye are limited. One approach that has gained increased promise is high-resolution retinal imaging that has undergone tremendous technological advances over the last two decades to probe the living retina at the cellular level. Pivotal in these advances has been adaptive optics (AO) and optical coherence tomography (OCT) that together allow unprecedented spatial resolution of retinal structures in all three dimensions. Using these high-resolution systems, cone photoreceptor are now routinely imaged in healthy and diseased retina enabling fundamental structural properties of cones to be studied such as cell spacing, packing arrangement, and alignment. Other important cell properties, however, have remained elusive to investigation as even better imaging performance is required and thus has resulted in an incomplete understanding of how cells in the photoreceptor-RPE complex interact with light. To address this technical bottleneck, we expanded the imaging capability of AO-OCT to detect and quantify more accurately and completely the optical properties of cone photoreceptor and RPE cells at the cellular level in the living human retina. The first objective of this thesis was development of a new AO-OCT method that is more precise and sensitive, thus enabling a more detailed view of the 3D optical signature of the photoreceptor-RPE complex than was previously possible (Chapter 2). Using this new system, the second objective was quantifying the waveguide properties of individual cone photoreceptor inner and outer segments across the macula (Chapter 3). The third objective extended the AO

  14. Photoreceptor effects on plant biomass, resource allocation, and metabolic state.

    Science.gov (United States)

    Yang, Deyue; Seaton, Daniel D; Krahmer, Johanna; Halliday, Karen J

    2016-07-05

    Plants sense the light environment through an ensemble of photoreceptors. Members of the phytochrome class of light receptors are known to play a critical role in seedling establishment, and are among the best-characterized plant signaling components. Phytochromes also regulate adult plant growth; however, our knowledge of this process is rather fragmented. This study demonstrates that phytochrome controls carbon allocation and biomass production in the developing plant. Phytochrome mutants have a reduced CO2 uptake, yet overaccumulate daytime sucrose and starch. This finding suggests that even though carbon fixation is impeded, the available carbon resources are not fully used for growth during the day. Supporting this notion, phytochrome depletion alters the proportion of day:night growth. In addition, phytochrome loss leads to sizeable reductions in overall growth, dry weight, total protein levels, and the expression of CELLULOSE SYNTHASE-LIKE genes. Because cellulose and protein are major constituents of plant biomass, our data point to an important role for phytochrome in regulating these fundamental components of plant productivity. We show that phytochrome loss impacts core metabolism, leading to elevated levels of tricarboxylic acid cycle intermediates, amino acids, sugar derivatives, and notably the stress metabolites proline and raffinose. Furthermore, the already growth-retarded phytochrome mutants are less responsive to growth-inhibiting abiotic stresses and have elevated expression of stress marker genes. This coordinated response appears to divert resources from energetically costly biomass production to improve resilience. In nature, this strategy may be activated in phytochrome-disabling, vegetation-dense habitats to enhance survival in potentially resource-limiting conditions.

  15. The carcinine transporter CarT is required in Drosophila photoreceptor neurons to sustain histamine recycling.

    Science.gov (United States)

    Stenesen, Drew; Moehlman, Andrew T; Krämer, Helmut

    2015-12-14

    Synaptic transmission from Drosophila photoreceptors to lamina neurons requires recycling of histamine neurotransmitter. Synaptic histamine is cleared by uptake into glia and conversion into carcinine, which functions as transport metabolite. How carcinine is transported from glia to photoreceptor neurons remains unclear. In a targeted RNAi screen for genes involved in this pathway, we identified carT, which encodes a member of the SLC22A transporter family. CarT expression in photoreceptors is necessary and sufficient for fly vision and behavior. Carcinine accumulates in the lamina of carT flies. Wild-type levels are restored by photoreceptor-specific expression of CarT, and endogenous tagging suggests CarT localizes to synaptic endings. Heterologous expression of CarT in S2 cells is sufficient for carcinine uptake, demonstrating the ability of CarT to utilize carcinine as a transport substrate. Together, our results demonstrate that CarT transports the histamine metabolite carcinine into photoreceptor neurons, thus contributing an essential step to the histamine-carcinine cycle.

  16. The carcinine transporter CarT is required in Drosophila photoreceptor neurons to sustain histamine recycling

    Science.gov (United States)

    Stenesen, Drew; Moehlman, Andrew T; Krämer, Helmut

    2015-01-01

    Synaptic transmission from Drosophila photoreceptors to lamina neurons requires recycling of histamine neurotransmitter. Synaptic histamine is cleared by uptake into glia and conversion into carcinine, which functions as transport metabolite. How carcinine is transported from glia to photoreceptor neurons remains unclear. In a targeted RNAi screen for genes involved in this pathway, we identified carT, which encodes a member of the SLC22A transporter family. CarT expression in photoreceptors is necessary and sufficient for fly vision and behavior. Carcinine accumulates in the lamina of carT flies. Wild-type levels are restored by photoreceptor-specific expression of CarT, and endogenous tagging suggests CarT localizes to synaptic endings. Heterologous expression of CarT in S2 cells is sufficient for carcinine uptake, demonstrating the ability of CarT to utilize carcinine as a transport substrate. Together, our results demonstrate that CarT transports the histamine metabolite carcinine into photoreceptor neurons, thus contributing an essential step to the histamine–carcinine cycle. DOI: http://dx.doi.org/10.7554/eLife.10972.001 PMID:26653853

  17. Effects of Ranibizumab and Aflibercept on Human Müller Cells and Photoreceptors under Stress Conditions

    Science.gov (United States)

    Shen, Weiyong; Yau, Belinda; Lee, So-Ra; Zhu, Ling; Yam, Michelle; Gillies, Mark C.

    2017-01-01

    Anti-vascular endothelial growth factor (VEGF) therapy has revolutionized the treatment of retinal vascular diseases. However, constitutive VEGF also acts as a trophic factor on retinal non-vascular cells. We have studied the effects of aflibercept and ranibizumab on human Müller cells and photoreceptors exposed to starvation media containing various concentrations of glucose, with or without CoCl2-induced hypoxia. Cell survival was assessed by calcein-AM cell viability assays. Expression of heat shock proteins (Hsp) and redox proteins thioredoxin 1 and 2 (TRX1, TRX2) was studied by Western blots. The production of neurotrophic factors in Müller cells and interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors was measured by enzyme-linked immunosorbent assays. Aflibercept and ranibizumab did not affect the viability of both types of cells. Neither aflibercept nor ranibizumab affected the production of neurotrophic factors or expression of Hsp60 and Hsp90 in Müller cells. However, aflibercept but not ranibizumab affected the expression of Hsp60, Hsp9, TRX1 and TRX2 in photoreceptors. Aflibercept and ranibizumab both inhibited the production of IRBP in photoreceptors, aflibercept more so than ranibizumab. Our data indicates that the potential influence of aflibercept and ranibizumab on photoreceptors should be specifically monitored in clinical studies. PMID:28257068

  18. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells

    Directory of Open Access Journals (Sweden)

    Yukari Komuta

    2016-06-01

    Full Text Available Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

  19. Ciliopathy-associated IQCB1/NPHP5 protein is required for mouse photoreceptor outer segment formation.

    Science.gov (United States)

    Ronquillo, Cecinio C; Hanke-Gogokhia, Christin; Revelo, Monica P; Frederick, Jeanne M; Jiang, Li; Baehr, Wolfgang

    2016-10-01

    Null mutations in the human IQCB1/NPHP5 (nephrocystin-5) gene that encodes NPHP5 are the most frequent cause of Senior-Løken syndrome, a ciliopathy that is characterized by Leber congenital amaurosis and nephronophthisis. We generated germline Nphp5-knockout mice by placing a β-Geo gene trap in intron 4, thereby truncating NPHP5 at Leu87 and removing all known functional domains. At eye opening, Nphp5(-/-) mice exhibited absence of scotopic and photopic electroretinogram responses, a phenotype that resembles Leber congenital amaurosis. Outer segment transmembrane protein accumulation in Nphp5(-/-) endoplasmic reticulum was evident as early as postnatal day (P)6. EGFP-CETN2, a centrosome and transition zone marker, identified basal bodies in Nphp5(-/-) ph